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Thiamine tetrahydrofurfuryl disulfide improves energy metabolism and physical performance during physical-fatigue loading in rats
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Nutrition Research 29 (2009) 867 – 872 www.nrjournal.com
Thiamine tetrahydrofurfuryl disulfide improves energy metabolism and physical performance during physical-fatigue loading in rats☆ Satoshi Nozaki a,c,d , Hiroshi Mizuma a,c,e , Masaaki Tanaka a,b,c , Guanghua Jin a,c,f , Tsuyoshi Tahara a,c,e , Kei Mizuno a,c,d , Masanori Yamato a,c,f , Kaori Okuyama a,c,f , Asami Eguchi a,c,f , Kouji Akimoto g , Takahito Kitayoshi g , Noriko Mochizuki-Oda h , Yosky Kataoka a,c,f,⁎, Yasuyoshi Watanabe a,c,d a Department of Physiology, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan Department of Biomarker and Molecular Biophysics, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan c The 21st Century Center of Excellence (COE) Program “Base to Overcome Fatigue” (from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government), Osaka 545-8585, Japan d Molecular Probe Dynamics Laboratory, Kobe, Hyogo 650-0047, Japan e Functional Probe Research Laboratory, Kobe, Hyogo 650-0047, Japan f Cellular Function Imaging Laboratory, RIKEN Center for Molecular Imaging Science, Kobe, Hyogo 650-0047, Japan g Consumer Healthcare Company, Health Science Laboratories, Takeda Pharmaceutical Company Limited, Osaka 532-8686, Japan h Department of Neurosurgery, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan Received 26 August 2009; revised 3 October 2009; accepted 12 October 2009 b
Abstract Impaired energy metabolism is considered a possible cause of fatigue. The thiamine derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is prescribed and is also an over-the-counter drug for the attenuation of fatigue. It is readily absorbed from the intestinal tract and converted into thiamine pyrophosphate (TPP), which plays an important role as a cofactor for enzymes of metabolic pathways involved in the production of adenosine triphosphate (ATP). We postulated that TTFD has an anti-fatigue effect by improving energy metabolism during physical-fatigue loading. Here, we initially used the forced swimming test to determine whether daily TTFD or thiamine for 5 days has anti-fatigue effects on weight-loaded rats. The swimming duration of TTFD-, but not of thiaminetreated rats, was significantly longer than that of control rats (P b .05). Based on these findings, we examined changes in the levels of thiamine and its phosphate esters in various organs and the effect of TTFD on ATP levels in skeletal muscle after forced swimming, to determine the cellular mechanisms of the anti-fatigue effect of TTFD. Daily TTFD resulted in a characteristic distribution of thiamine and its phosphate esters in rat skeletal muscle, liver, kidney, heart, brain, and plasma. Furthermore, daily TTFD attenuated the decrease in ATP content in the skeletal muscle caused by forced swimming with a weight load for a defined period (150 s). These results indicate that TTFD exerts anti-fatigue effects by improving energy metabolism during physical fatigue. © 2009 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
Adenosine triphosphate; Karoshi, Energy metabolism, Fatigue, Rat ATP, adenosine triphosphate; HPA, hypothalamo-pituitary-adrenal; HPLC, high-performance liquid chromatography; SEM, standard error of the mean; TCA, tricarboxylic acid; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate; TTFD, thiamine tetrahydrofurfuryl disulfide; TTP, thiamine triphosphate.
☆ Conflict of interest: Kouji Akimoto and Takahito Kitayoshi are employees of Takeda Pharmaceutical Co. Ltd., whose products were included in this study. Satoshi Nozaki, Hiroshi Mizuma, Masaaki Tanaka, Guanghua Jin, Tsuyoshi Tahara, Kei Mizuno, Masanori Yamato, Kaori Okuyama, Asami Eguchi, Yosky Kataoka and Yasuyoshi Watanabe declare no financial, general, or institutional competing interests. ⁎ Corresponding author. Cellular Function Imaging Laboratory, RIKEN Center for Molecular Imaging Science, Kobe, Hyogo 650-0047, Japan. Tel.: +81 78 304 7115; fax: +81 78 304 7161. E-mail address: [email protected] (Y. Kataoka).
0271-5317/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2009.10.007
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1. Introduction Fatigue is common in both sick and healthy individuals [1,2]. Chronic or accumulated fatigue can affect performance, and long-term accumulated fatigue can lead to karoshi (death as a result of overwork). Interest in the use of supplements for the prevention or attenuation of fatigue has recently increased. However, few studies have investigated the mechanism of anti-fatigue drugs, which has hindered the development of appropriate treatments for fatigue. We recently established a novel test of induced physical fatigue [3] and used it to demonstrate that the supplements, applephenon [4], coenzyme Q10 [5] and crocetin [6], attenuate physical fatigue. Physical fatigue can be derived from skeletal muscle action, which is also known as peripheral fatigue. Several reports have described the biochemical mechanisms of physical fatigue. For instance, it is associated with the depletion of physical energy sources such as glycogen, phosphocreatine and ATP [7]; a decrease in resting membrane potential or dysfunction of the calcium pump in the sarcoplasmic reticulum in the skeletal muscles; and the failure of neuromuscular transmission [8]. Reduced glucose (or energy) utilization has also been demonstrated [9] in a new animal model of fatigue [10]. Based on the aforementioned information, impaired energy metabolism appears to be associated with the pathophysiology of fatigue. Various intracellular processes generate ATP through glycolysis, the TCA cycle and the electron transport chain in the cytoplasm and mitochondria. Some thiamine-dependent enzymes are also involved in the pathway of ATP synthesis. A decrease in intracellular thiamine reduces enzymatic activity [11], which decreases ATP biosynthesis and might induce fatigue. Thiamine exists in its free form (T) but when absorbed from the digestive tract, it is converted to the phosphorylated forms, thiamine monophosphate (TMP), thiamine pyrophosphate (TPP), and thiamine triphosphate (TTP) [12]. The active form of thiamine is TPP, which serves as a cofactor for the enzymes involved in glucose metabolism; namely, transketolase, a constituent enzyme of the pentose-phosphate pathway, as well as the mitochondrial enzymes, pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase [13,14]. Hence, we hypothesize that thiamine should be considered as an anti-fatigue vitamin. The disulfide derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is a synthetic counterpart of allithiamine (a naturally occurring substance in garlic). The disulfide component renders TTFD more absorbent compared to readily-available water-soluble thiamine salts because it does not require the rate-limiting transport system required for thiamine absorption [15]. In addition, after uptake into various organs, TTFD is rapidly converted into various thiamine phosphate esters through thiamine in the cytosol [16]. Therefore, TTFD may be a more effective over-thecounter alternative to water-soluble thiamine salts for attenuating fatigue.
Tasks that cause physical fatigue are thought to consume intracellular thiamine phosphate esters and reduce the activities of thiamine-dependent enzymes involved in ATP biosynthesis. In fact, the activities of pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex in the brain and liver are depressed in thiamine-deficient rats [11]. We postulated that TTFD exerts anti-fatigue effects through improving ATP biosynthesis during physical (muscular) fatigue. Thiamine supplementation prevents exercise-induced fatigue in humans [17], although the mechanism has remained unclear. Following our hypothesis, our objective was to measure forced swimming duration in weight-loaded rats and to evaluate changes in the metabolism of thiamine derivatives and ATP in several tissues after the administration of TTFD or thiamine. During the forced swimming test without weight loading, the rats had a few breaks along the way where they float. Therefore, the forced swimming test without weight loading could not result in intensive exercise (personal communication). Consequently, the rats underwent weightloaded forced swimming tests. 2. Methods and materials 2.1. Chemicals and reagents Thiamine and TTFD were provided by Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). The standards for HPLC analysis were TMP purchased from Sigma (St. Louis, MO, USA), and TPP and TTP purchased from Wako Pure Chemical (Osaka, Japan). We obtained ATP from Sigma, and luciferin, luciferase, trichloroacetic acid, methanol (HPLC grade) and other chemicals from Wako Pure Chemical. 2.2. Experimental design We intraperitoneally administered saline, thiamine or TTFD to rats for 5 days and then evaluated anti-fatigue effects using the weight-loaded forced swimming test with some modifications [19]. To clarify the mechanisms of the anti-fatigue effects of TTFD, we determined the concentrations of thiamine and its phosphate esters in skeletal muscle, as well as the liver, kidney, heart, brain and plasma. Levels of ATP in the gastrocnemius and quadriceps femoris muscles of rats were compared after forced swimming for 150 s or no forced swimming. 2.3. Animals The Ethics for Animals Committee at Osaka City University Graduate School of Medicine approved the study protocol. Male Sprague-Dawley rats (7 weeks old) were housed in cages in a controlled environment at a temperature of 23 ± 1°C with 50 ± 5% humidity on a 12h light/dark schedule (lights on: 8:00 to 20:00). Food and water were available ad libitum. The composition of the CE2 (CLEA Japan Inc.) diet was (g/kg): carbohydrate (500),
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protein (251), fat (48), fiber (42) and water (93) with vitamin and mineral premixes. The rats were intraperitoneally injected with saline, thiamine (50 mg/kg) or TTFD (50 mg/kg), once each day for 5 days before evaluation of the anti-fatigue effects, since the precise determination of the effects of thiamine derivatives required their intracellular storage [16]. Thiamine or TTFD was dissolved in physiological saline and then adjusted to pH 6.5 using 1 N NaOH. The doses of thiamine and TTFD injected into the rats were determined as previously described [18]. The rats were euthanized by inhalation of an excess of diethyl ether after the experiments were concluded. 2.4. Weight-loaded forced swimming test The rats performed weight-loaded forced swimming tests with some modifications [19]. Briefly, steel rings weighing about 8% of the rat body weight were attached to the tail of each rat, which was then placed in a cylinder constructed of Plexiglas (18-cm inside diameter; 60-cm height) and containing water to a height of 40 cm. The temperature of the water in the cylinder was maintained at 23 ± 1°C. The time from the start of swimming with the weight to the point at which the rat could not return to the water surface for 10 s after sinking was measured. The rat was then removed from the cylinder and returned to its cage for recovery.
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min, the supernatant was passed through a 0.45-μm pore-size membrane filter (Millipore Corp., Tokyo, Japan). A portion of the filtrate was immediately frozen and stored at -80°C. The HPLC system comprised two pumps (LC-10Ai; Shimadzu Co. Ltd., Kyoto, Japan), a system controller (SCL-10Avp; Shimadzu Co.), a degasser (DGU-12A; Shimadzu Co.), a column oven (CTO-10Avp; Shimadzu Co.), a reaction system (S-3810; Soma Kogaku, Tokyo, Japan), an auto-injector (SIL-10Ai; Shimadzu Co.) and a Cosmosil 5C18-MS-II reversed-phase column (4.6 mm I.D. × 250 mm length; Nacalai Tesque, Kyoto, Japan) with a Cosmosil 5C18-MS-II guard cartridge column (4.6 mm I.D. × 100 mm length; Nacalai Tesque) coupled with a fluorometric detector (RF-10AxL; Shimadzu Co.). The excitation and emission wavelengths were 375 and 450 nm, respectively. The temperatures of the column and reaction coil were 40 and 50°C, respectively. The aqueous mobile phase comprised 0.2 mol/L of NaH2PO4 in 0.3% acetonitrile. The flow rate was maintained at 1.0 mL/min. Thiamine and its phosphate esters were derivatized after column separation into fluorophores in the reaction system using 15% NaOH, and 0.05% K3Fe(CN)6 at 50°C. The reaction coil consisted of a peak tube (0.45 μm I.D. × 6 m length) with the flow rate being maintained at 1.0 mL/min. The concentrations of thiamine and its phosphate esters were determined by calculating the areas under the curves using an integrator (LC-solution, Shimadzu Co.).
2.5. Sample collection and storage Levels of thiamine and its phosphate esters were determined as follows. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and then whole blood samples were collected from the abdominal aorta into tubes containing the anticoagulant disodium EDTA (1 mg/ml). The samples were deproteinized with 5% (v/v) trichloroacetic acid for 20 min on ice, centrifuged at 20,000 × g at 4°C for 10 min, and then the supernatant was stored at -80°C. The hindlimb muscles (gastrocnemius and quadriceps femoris), liver, kidney, heart, and brain were removed after perfusion with ice-cold saline. All tissues were immediately frozen in liquid nitrogen and stored at -80°C. After forced swimming for 150 s, levels of ATP were measured. The rats were immediately anesthetized with diethyl ether, and then the gastrocnemius and quadriceps femoris muscles were perfused with ice-cold saline, removed, rapidly frozen in liquid nitrogen and stored at -80°C. 2.6. Measurements of thiamine and its phosphate esters from blood and tissues The concentrations of thiamine and its phosphate esters were measured by HPLC as described by Kimura et al [20] with minor modifications. Skeletal muscle, liver, kidney, heart and brain was thawed, homogenized in 5 volumes of 10% (v/v) trichloroacetic acid, and then deproteinized for 20 min on ice. After centrifugation at 20,000 × g at 4°C for 20
2.7. Measurement of ATP levels The ATP contents were measured in the gastrocnemius and quadriceps femoris hindlimb muscles that were collected after weight-loaded forced swimming for 150 s. We previously assessed the association between swimming duration and dropout rates in the weight-loaded forced swimming test and found that about 80% of intact animals could swim for over 150 s in this test (N = 18, data not shown). Therefore, we defined the weight-loaded forced swimming duration as 150 s. According to the method of ATP measurements described by Mochizuki-Oda et al [21], tissues were homogenized in 1 mmol/L disodium EDTA containing 0.5 mol/L HClO4, centrifuged at 12,000 × g at 4°C for 15 min, and then the supernatant was neutralized with 1 N KOH. The KClO4 precipitate was removed by centrifugation of 12,000 × g at 4°C for 15 min, and the supernatant was mixed with 1/10 volume of 1 mol/L Tris–HCl (pH 7.5) and stored at -20°C for ATP assays. The precipitate denatured by HClO4 was dissolved in 0.5 N NaOH and used for quantitative protein analysis using the Bradford method [22]. We assayed ATP using the luciferase-luciferin method. 2.8. Statistical analyses Data are presented as means ± SEM, unless stated otherwise. All statistical analyses were performed using JMP software version 7.0 (SAS Institute Inc., Cary, NC). The
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significance of differences among the 3 groups was evaluated using the one-way analysis of variance (ANOVA). When statistically significant effects were found, intergroup differences between the control groups and those injected with thiamine or TTFD were evaluated using Dunnett's test. Changes in ATP levels between the swimming and non-swimming groups with daily administration of saline or TTFD were tested by two-way ANOVA followed by Student's two-tailed t-test. A probability level of P b .05 was considered statistically significant. 3. Results 3.1. Weight-loaded forced swimming test We initially measured the swimming duration of rats given thiamine (50 mg/kg, i.p.) or TTFD (50 mg/kg, i.p.). However, single injections of either one at 1, 3, 6, or 24 h before the weight-loaded forced swimming test had no apparent effect on swimming duration compared to that of the control group (data not shown). The administration of thiamine for 5 days did not significantly increase the swimming duration of the group injected (1.2-fold compared to control; P = .115);however, the swimming duration of the rats injected with TTFD for 5 days was significantly longer than that of the control group (1.7-fold compared to control; P b .05; Fig. 1). 3.2. Content of thiamine and its phosphate esters The effects of thiamine or TTFD administration on the concentrations of thiamine and its phosphate derivatives in rat skeletal muscle, liver, kidney, heart, brain and plasma are shown in Table 1. Concentrations of thiamine, TMP and TPP were higher in most of the organs from the rats given TTFD compared to those given thiamine. Thiamine increased TMP (1.3-fold compared to control; P b .05) and TPP (1.7-fold compared to control, P b .01) levels in the skeletal muscle;
Fig. 1. Effects of thiamine or thiamine tetrahydrofurfuryl disulfide on duration of forced swimming by weight-loaded rats. Rats were injected intraperitoneally with saline (Control), thiamine (Thiamine; 50 mg/kg), or with thiamine tetrahydrofurfuryl disulfide (TTFD; 50 mg/kg) once each day for 5 days. Values are means ± SEM (Control, n = 8; thiamine, n = 6; TTFD, n = 8). Data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's test. *P b .05, significantly different from corresponding control values.
Table 1 Effects of thiamine and thiamine tetrahydrofurfuryl disulfide administration on the concentrations of thiamine and its phosphate esters in several organs and plasma Saline (n = 8) Skeletal muscle Thiamine 0.06 ± 0.02 TMP 2.2 ± 0.2 TPP 3.2 ± 0.2 TTP 1.16 ± 0.06 Liver Thiamine 0.7 ± 0.1 TMP 2.3 ± 0.3 TPP 16.1 ± 1.2 TTP 1.2 ± 0.1 Kidney Thiamine 0.9 ± 0.1 TMP 2.6 ± 0.1 TPP 6.5 ± 0.2 TTP ND Heart Thiamine 1.40 ± 0.08 TMP 2.4 ± 0.3 TPP 10.9 ± 0.4 TTP 0.37 ± 0.06 Brain Thiamine 0.25 ± 0.01 TMP 2.6 ± 0.1 TPP 4.5 ± 0.1 TTP 0.47 ± 0.02 Plasma Thiamine 0.12 ± 0.01 TMP 0.35 ± 0.02 TPP 0.32 ± 0.01 TTP 0.071 ± 0.004
Thiamine (n = 6)
TTFD (n = 8)
0.08 ± 0.02 3.2 ± 0.1* 5.4 ± 0.4** 1.15 ± 0.06
0.24 ± 0.05** 3.6 ± 0.3** 7.3 ± 0.5*** 1.18 ± 0.05
1.5 ± 0.1 2.6 ± 0.3 16.7 ± 1.1 1.0 ± 0.1
5.0 ± 0.8** 3.5 ± 0.8 20.3 ± 1.1* 1.0 ± 0.1
1.6 ± 0.2 2.7 ± 0.1 6.8 ± 0.2 ND
5.2 ± 1.2** 3.1 ± 0.1** 7.1 ± 0.2 ND
1.28 ± 0.10 3.2 ± 0.6 11.1 ± 0.3 0.37 ± 0.03
1.54 ± 0.04 2.5 ± 0.2 14.1 ± 0.5** 0.37 ± 0.02
0.30 ± 0.01 2.8 ± 0.1 4.6 ± 0.1 0.34 ± 0.02
0.36 ± 0.02*** 2.8 ± 0.1 4.7 ± 0.1 0.29 ± 0.01***
0.18 ± 0.03 0.45 ± 0.03 0.37 ± 0.01 0.084 ± 0.009
0.69 ± 0.14** 0.61 ± 0.04** 0.83 ± 0.18** 0.081 ± 0.007
TTFD, thiamine tetrahydrofurfuryl disulfide; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate; TTP, thiamine triphosphate; ND, not detected. Rats were intraperitoneally injected with saline (Control), thiamine (50 mg/ kg, Thiamine), or thiamine tetrahydrofurfuryl disulfide (50 mg/kg, TTFD) once a day for 5 days. The values are means ± SEM (nmol/g wet tissue). ***P b .001, **P b .01, *P b .05, significantly different from the corresponding values of the saline-treated group. One-way analysis of variance was used to evaluate the significance of the difference among the 3 groups. When statistically significant effects were found, intergroup differences between the control and thiamine or TTFD injected groups were evaluated using Dunnett's test.
whereas, TTFD increased thiamine (4.0-fold compared to control, P b .01), TMP (1.6-fold compared to control, P b .01) and TPP (2.3-fold compared to control, P b .01) levels. We also found that TTFD increased levels of thiamine and TPP in the liver, thiamine and TMP in the kidney, TPP and thiamine in the heart, and TMP and TPP in plasma. By contrast, data in Table 1 shows that brain TTP levels decreased in rats given thiamine or TTFD (P b .001). 3.3. Muscle ATP levels The effect of TTFD on muscle ATP levels of rats after weight-loaded forced swimming for 150 s was examined. Forced swimming decreased ATP levels in the gastrocne-
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mius of rats injected with saline (vehicle) for 5 days (27 ± 10%; compared to the non swimming group), but exerted a significantly smaller decrease in those of rats injected with TTFD for 5 days (14 ± 15% reduction compared to the nonswimming group). The tendency was the same in the quadriceps femoris: the ATP level was decreased in the quadriceps femoris of the groups injected with saline or with TTFD by 7 ± 2% and by 2 ± 7%, respectively, compared to the non-swimming group.
4. Discussion The biochemical mechanisms of physical or peripheral fatigue generated through skeletal muscle actions have been described. Peripheral fatigue is associated with the depletion of glycogen, phosphocreatine, and ATP [7]; a decrease in the resting membrane potential or dysfunction of the calcium pump in the sarcoplasmic reticulum in the skeletal muscles; and failure of neuromuscular transmission [8]. Thus, an impaired energy state might contribute to physical fatigue. The decrease in the ATP level is larger in the gastrocnemius compared to the other three hindlimb muscles after forced swimming with 5% body weight-loading in rats [7], findings with which the present study agrees. Forced swimming for 150 s decreased the ATP level in the gastrocnemius by 27% and in the quadriceps femoris by 7%. This large difference might be due to the heavier load on the gastrocnemius than on the quadriceps femoris during forced swimming. Muscular contraction during anaerobic exercise initially generates ATP from phosphocreatine, and then ATP is subsequently depleted [23,24]. After phosphocreatine is consumed, high ATP utilization in skeletal muscle leads to ATP production not only through muscular glycogenolysis [24], but also through the utilization of blood free glucose generated from hepatic glycogen [25]. Our preliminary study showed that the plasma glucose level increases after the end of forced swimming, and that the increase in the glucose concentration reaches a maximum at 5 min after the end of the swimming. The plasma glucose concentration would increase due to activation of the hypothalamo-pituitaryadrenal (HPA) axis. The present results indicate that TTFD accelerates ATP re-synthesis by accumulating high levels of TPP and facilitates glycogenolysis in the skeletal muscle during exercise, resulting in attenuation of the exerciseinduced decrease in muscle ATP levels. A limitation associated with this study is the small sample number. The muscle ATP levels varied considerably among individual rats. Studies using more animals are essential to generalize our results. One study of oarsmen who consumed 4,000 kcal (or more) per day also consumed more than the recommended amount of thiamine; yet, most of them were thiaminedeficient [26], which causes fatigue [27,28]. Thus, the recovery of the physical laborers and athletes from fatigue described by Nakamura and colleagues might have been due
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to thiamine supplementation that addressed a thiamine deficiency [29]. The present study demonstrated that appropriate doses of thiamine generated by the daily consumption of TTFD could improve physical performance. Therefore, daily TTFD supplementation might serve as a novel strategy to prevent or attenuate fatigue in humans. Acknowledgment This study was supported in part by the 21st Century Center of Excellence (COE) Program, “Base to Overcome Fatigue” from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. The authors thank Norma Foster for editorial help with the manuscript. References [1] Ream E, Richardson A. Fatigue in patients with cancer and chronic obstructive airways disease: a phenomenological enquiry. Int J Nurs Stud 1997;34:44-53. [2] Grandjean EP. Fatigue. Am Ind Hyg Assoc J 1970;31:401-11. [3] Nozaki S, Tanaka M, Mizuno K, Ataka S, Mizuma H, Tahara T, et al. Mental and physical fatigue-related biochemical alterations. Nutrition 2009;25:51-7. [4] Ataka S, Tanaka M, Nozaki S, Mizuma H, Mizuno K, Tahara T, et al. Effects of applephenon and ascorbic acid on physical fatigue. Nutrition 2007;23:419-23. [5] Mizuno K, Tanaka M, Nozaki S, Mizuma H, Ataka S, Tahara T, et al. Antifatigue effects of coenzyme Q10 during physical fatigue. Nutrition 2008;24:293-9. [6] Mizuma H, Tanaka M, Nozaki S, Mizuno K, Tahara T, Ataka S, et al. Daily oral administration of crocetin attenuates physical fatigue in human. Nutr Res 2009;29:145-50. [7] Lindinger MI, Heigenhauser GJ, Spriet LL. Effects of intense swimming and titanicelectrical stimulation on skeletal muscle ions and metabolites. J Appl Physiol 1987;63:2331-9. [8] Fitts RH. Cellular mechanisms of muscle fatigue. Physiol Rev 1994; 74:49-94. [9] Tanaka M, Nakamura F, Mizokawa S, Matsumura A, Nozaki S, Watanabe Y. Establishment and assessment of a rat model of fatigue. Neurosci Lett 2003;352:159-62. [10] Tanaka M, Watanabe Y. Reduced energy utilization in the brain is a feature of an animal model of fatigue. Int J Neurosci 2008;118:683-92. [11] Parker Jr WD, Haas R, Stumpf DA, Parks J, Eguren LA, Jackson C. Brain mitochondrial metabolism in experimental thiamine deficiency. Neurology 1984;34(11):1477-81. [12] Batifoulier F, Verny MA, Besson C, Demigne C, Remesy C. Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column. J Chromatogr B Analyt Technol Biomed Life Sci 2005;816:67-72. [13] Gaitonde MK, Fayein NA, Johnson AL. Decreased metabolism in vivo of glucose into amino acids of the brain of thiamine-deficient rats after treatment with pyrithiamine. J Neurochem 1975;24:1215-23. [14] Hollowach J, Kauffman F, Ikossi MG, Thomas C, McDougal DGJ. The effects of a thiamine antagonist, pyrithiamine, on levels of selected metabolic intermediates and on activities of thiamine-dependent enzymes in the brain and liver. J Neurochem 1968;15:621-31. [15] Shimazono N, Katsura E. Beriberi and Thiamine. Tokyo: Igaku Shoin Ltd.; 1965. [16] Sen I, Cooper JR. The turnover of thiamine and its phosphate esters in rat organs. Neurochem Res 1976;1:65-71. [17] Suzuki M, Itokawa Y. Effects of thiamine supplementation on exercise-induced fatigue. Metab Brain Dis 1996;11(1):95–106.
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Nutrition Research 29 (2009) 867 – 872 www.nrjournal.com
Thiamine tetrahydrofurfuryl disulfide improves energy metabolism and physical performance during physical-fatigue loading in rats☆ Satoshi Nozaki a,c,d , Hiroshi Mizuma a,c,e , Masaaki Tanaka a,b,c , Guanghua Jin a,c,f , Tsuyoshi Tahara a,c,e , Kei Mizuno a,c,d , Masanori Yamato a,c,f , Kaori Okuyama a,c,f , Asami Eguchi a,c,f , Kouji Akimoto g , Takahito Kitayoshi g , Noriko Mochizuki-Oda h , Yosky Kataoka a,c,f,⁎, Yasuyoshi Watanabe a,c,d a Department of Physiology, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan Department of Biomarker and Molecular Biophysics, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan c The 21st Century Center of Excellence (COE) Program “Base to Overcome Fatigue” (from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government), Osaka 545-8585, Japan d Molecular Probe Dynamics Laboratory, Kobe, Hyogo 650-0047, Japan e Functional Probe Research Laboratory, Kobe, Hyogo 650-0047, Japan f Cellular Function Imaging Laboratory, RIKEN Center for Molecular Imaging Science, Kobe, Hyogo 650-0047, Japan g Consumer Healthcare Company, Health Science Laboratories, Takeda Pharmaceutical Company Limited, Osaka 532-8686, Japan h Department of Neurosurgery, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan Received 26 August 2009; revised 3 October 2009; accepted 12 October 2009 b
Abstract Impaired energy metabolism is considered a possible cause of fatigue. The thiamine derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is prescribed and is also an over-the-counter drug for the attenuation of fatigue. It is readily absorbed from the intestinal tract and converted into thiamine pyrophosphate (TPP), which plays an important role as a cofactor for enzymes of metabolic pathways involved in the production of adenosine triphosphate (ATP). We postulated that TTFD has an anti-fatigue effect by improving energy metabolism during physical-fatigue loading. Here, we initially used the forced swimming test to determine whether daily TTFD or thiamine for 5 days has anti-fatigue effects on weight-loaded rats. The swimming duration of TTFD-, but not of thiaminetreated rats, was significantly longer than that of control rats (P b .05). Based on these findings, we examined changes in the levels of thiamine and its phosphate esters in various organs and the effect of TTFD on ATP levels in skeletal muscle after forced swimming, to determine the cellular mechanisms of the anti-fatigue effect of TTFD. Daily TTFD resulted in a characteristic distribution of thiamine and its phosphate esters in rat skeletal muscle, liver, kidney, heart, brain, and plasma. Furthermore, daily TTFD attenuated the decrease in ATP content in the skeletal muscle caused by forced swimming with a weight load for a defined period (150 s). These results indicate that TTFD exerts anti-fatigue effects by improving energy metabolism during physical fatigue. © 2009 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
Adenosine triphosphate; Karoshi, Energy metabolism, Fatigue, Rat ATP, adenosine triphosphate; HPA, hypothalamo-pituitary-adrenal; HPLC, high-performance liquid chromatography; SEM, standard error of the mean; TCA, tricarboxylic acid; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate; TTFD, thiamine tetrahydrofurfuryl disulfide; TTP, thiamine triphosphate.
☆ Conflict of interest: Kouji Akimoto and Takahito Kitayoshi are employees of Takeda Pharmaceutical Co. Ltd., whose products were included in this study. Satoshi Nozaki, Hiroshi Mizuma, Masaaki Tanaka, Guanghua Jin, Tsuyoshi Tahara, Kei Mizuno, Masanori Yamato, Kaori Okuyama, Asami Eguchi, Yosky Kataoka and Yasuyoshi Watanabe declare no financial, general, or institutional competing interests. ⁎ Corresponding author. Cellular Function Imaging Laboratory, RIKEN Center for Molecular Imaging Science, Kobe, Hyogo 650-0047, Japan. Tel.: +81 78 304 7115; fax: +81 78 304 7161. E-mail address: [email protected] (Y. Kataoka).
0271-5317/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2009.10.007
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1. Introduction Fatigue is common in both sick and healthy individuals [1,2]. Chronic or accumulated fatigue can affect performance, and long-term accumulated fatigue can lead to karoshi (death as a result of overwork). Interest in the use of supplements for the prevention or attenuation of fatigue has recently increased. However, few studies have investigated the mechanism of anti-fatigue drugs, which has hindered the development of appropriate treatments for fatigue. We recently established a novel test of induced physical fatigue [3] and used it to demonstrate that the supplements, applephenon [4], coenzyme Q10 [5] and crocetin [6], attenuate physical fatigue. Physical fatigue can be derived from skeletal muscle action, which is also known as peripheral fatigue. Several reports have described the biochemical mechanisms of physical fatigue. For instance, it is associated with the depletion of physical energy sources such as glycogen, phosphocreatine and ATP [7]; a decrease in resting membrane potential or dysfunction of the calcium pump in the sarcoplasmic reticulum in the skeletal muscles; and the failure of neuromuscular transmission [8]. Reduced glucose (or energy) utilization has also been demonstrated [9] in a new animal model of fatigue [10]. Based on the aforementioned information, impaired energy metabolism appears to be associated with the pathophysiology of fatigue. Various intracellular processes generate ATP through glycolysis, the TCA cycle and the electron transport chain in the cytoplasm and mitochondria. Some thiamine-dependent enzymes are also involved in the pathway of ATP synthesis. A decrease in intracellular thiamine reduces enzymatic activity [11], which decreases ATP biosynthesis and might induce fatigue. Thiamine exists in its free form (T) but when absorbed from the digestive tract, it is converted to the phosphorylated forms, thiamine monophosphate (TMP), thiamine pyrophosphate (TPP), and thiamine triphosphate (TTP) [12]. The active form of thiamine is TPP, which serves as a cofactor for the enzymes involved in glucose metabolism; namely, transketolase, a constituent enzyme of the pentose-phosphate pathway, as well as the mitochondrial enzymes, pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase [13,14]. Hence, we hypothesize that thiamine should be considered as an anti-fatigue vitamin. The disulfide derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is a synthetic counterpart of allithiamine (a naturally occurring substance in garlic). The disulfide component renders TTFD more absorbent compared to readily-available water-soluble thiamine salts because it does not require the rate-limiting transport system required for thiamine absorption [15]. In addition, after uptake into various organs, TTFD is rapidly converted into various thiamine phosphate esters through thiamine in the cytosol [16]. Therefore, TTFD may be a more effective over-thecounter alternative to water-soluble thiamine salts for attenuating fatigue.
Tasks that cause physical fatigue are thought to consume intracellular thiamine phosphate esters and reduce the activities of thiamine-dependent enzymes involved in ATP biosynthesis. In fact, the activities of pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex in the brain and liver are depressed in thiamine-deficient rats [11]. We postulated that TTFD exerts anti-fatigue effects through improving ATP biosynthesis during physical (muscular) fatigue. Thiamine supplementation prevents exercise-induced fatigue in humans [17], although the mechanism has remained unclear. Following our hypothesis, our objective was to measure forced swimming duration in weight-loaded rats and to evaluate changes in the metabolism of thiamine derivatives and ATP in several tissues after the administration of TTFD or thiamine. During the forced swimming test without weight loading, the rats had a few breaks along the way where they float. Therefore, the forced swimming test without weight loading could not result in intensive exercise (personal communication). Consequently, the rats underwent weightloaded forced swimming tests. 2. Methods and materials 2.1. Chemicals and reagents Thiamine and TTFD were provided by Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). The standards for HPLC analysis were TMP purchased from Sigma (St. Louis, MO, USA), and TPP and TTP purchased from Wako Pure Chemical (Osaka, Japan). We obtained ATP from Sigma, and luciferin, luciferase, trichloroacetic acid, methanol (HPLC grade) and other chemicals from Wako Pure Chemical. 2.2. Experimental design We intraperitoneally administered saline, thiamine or TTFD to rats for 5 days and then evaluated anti-fatigue effects using the weight-loaded forced swimming test with some modifications [19]. To clarify the mechanisms of the anti-fatigue effects of TTFD, we determined the concentrations of thiamine and its phosphate esters in skeletal muscle, as well as the liver, kidney, heart, brain and plasma. Levels of ATP in the gastrocnemius and quadriceps femoris muscles of rats were compared after forced swimming for 150 s or no forced swimming. 2.3. Animals The Ethics for Animals Committee at Osaka City University Graduate School of Medicine approved the study protocol. Male Sprague-Dawley rats (7 weeks old) were housed in cages in a controlled environment at a temperature of 23 ± 1°C with 50 ± 5% humidity on a 12h light/dark schedule (lights on: 8:00 to 20:00). Food and water were available ad libitum. The composition of the CE2 (CLEA Japan Inc.) diet was (g/kg): carbohydrate (500),
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protein (251), fat (48), fiber (42) and water (93) with vitamin and mineral premixes. The rats were intraperitoneally injected with saline, thiamine (50 mg/kg) or TTFD (50 mg/kg), once each day for 5 days before evaluation of the anti-fatigue effects, since the precise determination of the effects of thiamine derivatives required their intracellular storage [16]. Thiamine or TTFD was dissolved in physiological saline and then adjusted to pH 6.5 using 1 N NaOH. The doses of thiamine and TTFD injected into the rats were determined as previously described [18]. The rats were euthanized by inhalation of an excess of diethyl ether after the experiments were concluded. 2.4. Weight-loaded forced swimming test The rats performed weight-loaded forced swimming tests with some modifications [19]. Briefly, steel rings weighing about 8% of the rat body weight were attached to the tail of each rat, which was then placed in a cylinder constructed of Plexiglas (18-cm inside diameter; 60-cm height) and containing water to a height of 40 cm. The temperature of the water in the cylinder was maintained at 23 ± 1°C. The time from the start of swimming with the weight to the point at which the rat could not return to the water surface for 10 s after sinking was measured. The rat was then removed from the cylinder and returned to its cage for recovery.
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min, the supernatant was passed through a 0.45-μm pore-size membrane filter (Millipore Corp., Tokyo, Japan). A portion of the filtrate was immediately frozen and stored at -80°C. The HPLC system comprised two pumps (LC-10Ai; Shimadzu Co. Ltd., Kyoto, Japan), a system controller (SCL-10Avp; Shimadzu Co.), a degasser (DGU-12A; Shimadzu Co.), a column oven (CTO-10Avp; Shimadzu Co.), a reaction system (S-3810; Soma Kogaku, Tokyo, Japan), an auto-injector (SIL-10Ai; Shimadzu Co.) and a Cosmosil 5C18-MS-II reversed-phase column (4.6 mm I.D. × 250 mm length; Nacalai Tesque, Kyoto, Japan) with a Cosmosil 5C18-MS-II guard cartridge column (4.6 mm I.D. × 100 mm length; Nacalai Tesque) coupled with a fluorometric detector (RF-10AxL; Shimadzu Co.). The excitation and emission wavelengths were 375 and 450 nm, respectively. The temperatures of the column and reaction coil were 40 and 50°C, respectively. The aqueous mobile phase comprised 0.2 mol/L of NaH2PO4 in 0.3% acetonitrile. The flow rate was maintained at 1.0 mL/min. Thiamine and its phosphate esters were derivatized after column separation into fluorophores in the reaction system using 15% NaOH, and 0.05% K3Fe(CN)6 at 50°C. The reaction coil consisted of a peak tube (0.45 μm I.D. × 6 m length) with the flow rate being maintained at 1.0 mL/min. The concentrations of thiamine and its phosphate esters were determined by calculating the areas under the curves using an integrator (LC-solution, Shimadzu Co.).
2.5. Sample collection and storage Levels of thiamine and its phosphate esters were determined as follows. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and then whole blood samples were collected from the abdominal aorta into tubes containing the anticoagulant disodium EDTA (1 mg/ml). The samples were deproteinized with 5% (v/v) trichloroacetic acid for 20 min on ice, centrifuged at 20,000 × g at 4°C for 10 min, and then the supernatant was stored at -80°C. The hindlimb muscles (gastrocnemius and quadriceps femoris), liver, kidney, heart, and brain were removed after perfusion with ice-cold saline. All tissues were immediately frozen in liquid nitrogen and stored at -80°C. After forced swimming for 150 s, levels of ATP were measured. The rats were immediately anesthetized with diethyl ether, and then the gastrocnemius and quadriceps femoris muscles were perfused with ice-cold saline, removed, rapidly frozen in liquid nitrogen and stored at -80°C. 2.6. Measurements of thiamine and its phosphate esters from blood and tissues The concentrations of thiamine and its phosphate esters were measured by HPLC as described by Kimura et al [20] with minor modifications. Skeletal muscle, liver, kidney, heart and brain was thawed, homogenized in 5 volumes of 10% (v/v) trichloroacetic acid, and then deproteinized for 20 min on ice. After centrifugation at 20,000 × g at 4°C for 20
2.7. Measurement of ATP levels The ATP contents were measured in the gastrocnemius and quadriceps femoris hindlimb muscles that were collected after weight-loaded forced swimming for 150 s. We previously assessed the association between swimming duration and dropout rates in the weight-loaded forced swimming test and found that about 80% of intact animals could swim for over 150 s in this test (N = 18, data not shown). Therefore, we defined the weight-loaded forced swimming duration as 150 s. According to the method of ATP measurements described by Mochizuki-Oda et al [21], tissues were homogenized in 1 mmol/L disodium EDTA containing 0.5 mol/L HClO4, centrifuged at 12,000 × g at 4°C for 15 min, and then the supernatant was neutralized with 1 N KOH. The KClO4 precipitate was removed by centrifugation of 12,000 × g at 4°C for 15 min, and the supernatant was mixed with 1/10 volume of 1 mol/L Tris–HCl (pH 7.5) and stored at -20°C for ATP assays. The precipitate denatured by HClO4 was dissolved in 0.5 N NaOH and used for quantitative protein analysis using the Bradford method [22]. We assayed ATP using the luciferase-luciferin method. 2.8. Statistical analyses Data are presented as means ± SEM, unless stated otherwise. All statistical analyses were performed using JMP software version 7.0 (SAS Institute Inc., Cary, NC). The
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significance of differences among the 3 groups was evaluated using the one-way analysis of variance (ANOVA). When statistically significant effects were found, intergroup differences between the control groups and those injected with thiamine or TTFD were evaluated using Dunnett's test. Changes in ATP levels between the swimming and non-swimming groups with daily administration of saline or TTFD were tested by two-way ANOVA followed by Student's two-tailed t-test. A probability level of P b .05 was considered statistically significant. 3. Results 3.1. Weight-loaded forced swimming test We initially measured the swimming duration of rats given thiamine (50 mg/kg, i.p.) or TTFD (50 mg/kg, i.p.). However, single injections of either one at 1, 3, 6, or 24 h before the weight-loaded forced swimming test had no apparent effect on swimming duration compared to that of the control group (data not shown). The administration of thiamine for 5 days did not significantly increase the swimming duration of the group injected (1.2-fold compared to control; P = .115);however, the swimming duration of the rats injected with TTFD for 5 days was significantly longer than that of the control group (1.7-fold compared to control; P b .05; Fig. 1). 3.2. Content of thiamine and its phosphate esters The effects of thiamine or TTFD administration on the concentrations of thiamine and its phosphate derivatives in rat skeletal muscle, liver, kidney, heart, brain and plasma are shown in Table 1. Concentrations of thiamine, TMP and TPP were higher in most of the organs from the rats given TTFD compared to those given thiamine. Thiamine increased TMP (1.3-fold compared to control; P b .05) and TPP (1.7-fold compared to control, P b .01) levels in the skeletal muscle;
Fig. 1. Effects of thiamine or thiamine tetrahydrofurfuryl disulfide on duration of forced swimming by weight-loaded rats. Rats were injected intraperitoneally with saline (Control), thiamine (Thiamine; 50 mg/kg), or with thiamine tetrahydrofurfuryl disulfide (TTFD; 50 mg/kg) once each day for 5 days. Values are means ± SEM (Control, n = 8; thiamine, n = 6; TTFD, n = 8). Data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's test. *P b .05, significantly different from corresponding control values.
Table 1 Effects of thiamine and thiamine tetrahydrofurfuryl disulfide administration on the concentrations of thiamine and its phosphate esters in several organs and plasma Saline (n = 8) Skeletal muscle Thiamine 0.06 ± 0.02 TMP 2.2 ± 0.2 TPP 3.2 ± 0.2 TTP 1.16 ± 0.06 Liver Thiamine 0.7 ± 0.1 TMP 2.3 ± 0.3 TPP 16.1 ± 1.2 TTP 1.2 ± 0.1 Kidney Thiamine 0.9 ± 0.1 TMP 2.6 ± 0.1 TPP 6.5 ± 0.2 TTP ND Heart Thiamine 1.40 ± 0.08 TMP 2.4 ± 0.3 TPP 10.9 ± 0.4 TTP 0.37 ± 0.06 Brain Thiamine 0.25 ± 0.01 TMP 2.6 ± 0.1 TPP 4.5 ± 0.1 TTP 0.47 ± 0.02 Plasma Thiamine 0.12 ± 0.01 TMP 0.35 ± 0.02 TPP 0.32 ± 0.01 TTP 0.071 ± 0.004
Thiamine (n = 6)
TTFD (n = 8)
0.08 ± 0.02 3.2 ± 0.1* 5.4 ± 0.4** 1.15 ± 0.06
0.24 ± 0.05** 3.6 ± 0.3** 7.3 ± 0.5*** 1.18 ± 0.05
1.5 ± 0.1 2.6 ± 0.3 16.7 ± 1.1 1.0 ± 0.1
5.0 ± 0.8** 3.5 ± 0.8 20.3 ± 1.1* 1.0 ± 0.1
1.6 ± 0.2 2.7 ± 0.1 6.8 ± 0.2 ND
5.2 ± 1.2** 3.1 ± 0.1** 7.1 ± 0.2 ND
1.28 ± 0.10 3.2 ± 0.6 11.1 ± 0.3 0.37 ± 0.03
1.54 ± 0.04 2.5 ± 0.2 14.1 ± 0.5** 0.37 ± 0.02
0.30 ± 0.01 2.8 ± 0.1 4.6 ± 0.1 0.34 ± 0.02
0.36 ± 0.02*** 2.8 ± 0.1 4.7 ± 0.1 0.29 ± 0.01***
0.18 ± 0.03 0.45 ± 0.03 0.37 ± 0.01 0.084 ± 0.009
0.69 ± 0.14** 0.61 ± 0.04** 0.83 ± 0.18** 0.081 ± 0.007
TTFD, thiamine tetrahydrofurfuryl disulfide; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate; TTP, thiamine triphosphate; ND, not detected. Rats were intraperitoneally injected with saline (Control), thiamine (50 mg/ kg, Thiamine), or thiamine tetrahydrofurfuryl disulfide (50 mg/kg, TTFD) once a day for 5 days. The values are means ± SEM (nmol/g wet tissue). ***P b .001, **P b .01, *P b .05, significantly different from the corresponding values of the saline-treated group. One-way analysis of variance was used to evaluate the significance of the difference among the 3 groups. When statistically significant effects were found, intergroup differences between the control and thiamine or TTFD injected groups were evaluated using Dunnett's test.
whereas, TTFD increased thiamine (4.0-fold compared to control, P b .01), TMP (1.6-fold compared to control, P b .01) and TPP (2.3-fold compared to control, P b .01) levels. We also found that TTFD increased levels of thiamine and TPP in the liver, thiamine and TMP in the kidney, TPP and thiamine in the heart, and TMP and TPP in plasma. By contrast, data in Table 1 shows that brain TTP levels decreased in rats given thiamine or TTFD (P b .001). 3.3. Muscle ATP levels The effect of TTFD on muscle ATP levels of rats after weight-loaded forced swimming for 150 s was examined. Forced swimming decreased ATP levels in the gastrocne-
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mius of rats injected with saline (vehicle) for 5 days (27 ± 10%; compared to the non swimming group), but exerted a significantly smaller decrease in those of rats injected with TTFD for 5 days (14 ± 15% reduction compared to the nonswimming group). The tendency was the same in the quadriceps femoris: the ATP level was decreased in the quadriceps femoris of the groups injected with saline or with TTFD by 7 ± 2% and by 2 ± 7%, respectively, compared to the non-swimming group.
4. Discussion The biochemical mechanisms of physical or peripheral fatigue generated through skeletal muscle actions have been described. Peripheral fatigue is associated with the depletion of glycogen, phosphocreatine, and ATP [7]; a decrease in the resting membrane potential or dysfunction of the calcium pump in the sarcoplasmic reticulum in the skeletal muscles; and failure of neuromuscular transmission [8]. Thus, an impaired energy state might contribute to physical fatigue. The decrease in the ATP level is larger in the gastrocnemius compared to the other three hindlimb muscles after forced swimming with 5% body weight-loading in rats [7], findings with which the present study agrees. Forced swimming for 150 s decreased the ATP level in the gastrocnemius by 27% and in the quadriceps femoris by 7%. This large difference might be due to the heavier load on the gastrocnemius than on the quadriceps femoris during forced swimming. Muscular contraction during anaerobic exercise initially generates ATP from phosphocreatine, and then ATP is subsequently depleted [23,24]. After phosphocreatine is consumed, high ATP utilization in skeletal muscle leads to ATP production not only through muscular glycogenolysis [24], but also through the utilization of blood free glucose generated from hepatic glycogen [25]. Our preliminary study showed that the plasma glucose level increases after the end of forced swimming, and that the increase in the glucose concentration reaches a maximum at 5 min after the end of the swimming. The plasma glucose concentration would increase due to activation of the hypothalamo-pituitaryadrenal (HPA) axis. The present results indicate that TTFD accelerates ATP re-synthesis by accumulating high levels of TPP and facilitates glycogenolysis in the skeletal muscle during exercise, resulting in attenuation of the exerciseinduced decrease in muscle ATP levels. A limitation associated with this study is the small sample number. The muscle ATP levels varied considerably among individual rats. Studies using more animals are essential to generalize our results. One study of oarsmen who consumed 4,000 kcal (or more) per day also consumed more than the recommended amount of thiamine; yet, most of them were thiaminedeficient [26], which causes fatigue [27,28]. Thus, the recovery of the physical laborers and athletes from fatigue described by Nakamura and colleagues might have been due
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to thiamine supplementation that addressed a thiamine deficiency [29]. The present study demonstrated that appropriate doses of thiamine generated by the daily consumption of TTFD could improve physical performance. Therefore, daily TTFD supplementation might serve as a novel strategy to prevent or attenuate fatigue in humans. Acknowledgment This study was supported in part by the 21st Century Center of Excellence (COE) Program, “Base to Overcome Fatigue” from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. The authors thank Norma Foster for editorial help with the manuscript. References [1] Ream E, Richardson A. Fatigue in patients with cancer and chronic obstructive airways disease: a phenomenological enquiry. Int J Nurs Stud 1997;34:44-53. [2] Grandjean EP. Fatigue. Am Ind Hyg Assoc J 1970;31:401-11. [3] Nozaki S, Tanaka M, Mizuno K, Ataka S, Mizuma H, Tahara T, et al. Mental and physical fatigue-related biochemical alterations. Nutrition 2009;25:51-7. [4] Ataka S, Tanaka M, Nozaki S, Mizuma H, Mizuno K, Tahara T, et al. Effects of applephenon and ascorbic acid on physical fatigue. Nutrition 2007;23:419-23. [5] Mizuno K, Tanaka M, Nozaki S, Mizuma H, Ataka S, Tahara T, et al. Antifatigue effects of coenzyme Q10 during physical fatigue. Nutrition 2008;24:293-9. [6] Mizuma H, Tanaka M, Nozaki S, Mizuno K, Tahara T, Ataka S, et al. Daily oral administration of crocetin attenuates physical fatigue in human. Nutr Res 2009;29:145-50. [7] Lindinger MI, Heigenhauser GJ, Spriet LL. Effects of intense swimming and titanicelectrical stimulation on skeletal muscle ions and metabolites. J Appl Physiol 1987;63:2331-9. [8] Fitts RH. Cellular mechanisms of muscle fatigue. Physiol Rev 1994; 74:49-94. [9] Tanaka M, Nakamura F, Mizokawa S, Matsumura A, Nozaki S, Watanabe Y. Establishment and assessment of a rat model of fatigue. Neurosci Lett 2003;352:159-62. [10] Tanaka M, Watanabe Y. Reduced energy utilization in the brain is a feature of an animal model of fatigue. Int J Neurosci 2008;118:683-92. [11] Parker Jr WD, Haas R, Stumpf DA, Parks J, Eguren LA, Jackson C. Brain mitochondrial metabolism in experimental thiamine deficiency. Neurology 1984;34(11):1477-81. [12] Batifoulier F, Verny MA, Besson C, Demigne C, Remesy C. Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column. J Chromatogr B Analyt Technol Biomed Life Sci 2005;816:67-72. [13] Gaitonde MK, Fayein NA, Johnson AL. Decreased metabolism in vivo of glucose into amino acids of the brain of thiamine-deficient rats after treatment with pyrithiamine. J Neurochem 1975;24:1215-23. [14] Hollowach J, Kauffman F, Ikossi MG, Thomas C, McDougal DGJ. The effects of a thiamine antagonist, pyrithiamine, on levels of selected metabolic intermediates and on activities of thiamine-dependent enzymes in the brain and liver. J Neurochem 1968;15:621-31. [15] Shimazono N, Katsura E. Beriberi and Thiamine. Tokyo: Igaku Shoin Ltd.; 1965. [16] Sen I, Cooper JR. The turnover of thiamine and its phosphate esters in rat organs. Neurochem Res 1976;1:65-71. [17] Suzuki M, Itokawa Y. Effects of thiamine supplementation on exercise-induced fatigue. Metab Brain Dis 1996;11(1):95–106.
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