The Body Vitamin B 1 Levels of Rats Fed a Diet Containing the Minimum Requirement of Vitamin B 1 Is Reduced by Exercise

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1 J Nutr Si Vitminol, 59, 87 92, 213 The Body Vitmin B 1 Levels of Rts Fed Diet Contining the Minimum Requirement of Vitmin B 1 Is Redued y Exerise Ktsumi Shit nd Tsutomu Fukuwtri Deprtment of Food Siene nd Nutrition, Shool of Humn Cultures, The University of Shig Prefeture, Hikone, Shig , Jpn (Reeived Otoer 16, 212) Summry It is thought tht inresing energy expenditure inreses onsumption of vitmin B 1, leding to n inrese in the requirement of vitmin B 1. However, evidene supporting this hypothesis is lking. To exmine the hypothesis, initilly, we determined the minimum requirement of vitmin B 1 for wening rts. We found tht the minimum requirement of vitmin B 1 for optimum growth of wening rts ws round.786 mg thimin/kg diet. Next, rts fed diet ontining the minimum requirement of vitmin B 1 were fored to swim until exhustion. Conentrtions of vitmin B 1 in the lood nd liver s well s urinry exretion of swimming rts deresed signifintly ompred with those of nonswimming rts (p,.5), while in rts fed the diet ontining suffiient mount of vitmin B 1 (4.72 mg thimin/kg diet), vitmin B 1 mounts in the lood, liver nd urine were not ffeted y swimming. We lerly nd firstly showed the redution of ody vitmin B 1 following inreses in energy expenditure. Key Words vitmin B 1, requirement, exerise, rt, urine The ourrene of lssi thimin-defiieny syndromes suh s erieri is unommon in Jpn. However, mild-to-moderte defiienies of thimin n our in the elderly nd thletes (1, 2). Vitmin B 1 hs importnt roles in the enzyme retions involved in energy prodution. It is thought tht inresing energy expenditure inreses onsumption of vitmin B 1, leding to n inrese in the requirement of vitmin B 1. Nevertheless, evidene supporting this hypothesis is lking. For exmple, onentrtions of vitmin B 1 in lood, the liver nd urine were not deresed y intense exerise in rts fed purified diet (AIN-93) (3) reommended for growth. We thought tht the reson for this finding ws tht more thn the minimum requirement of vitmin B 1 hd een given. It is thought tht the mounts of vitmins in the AIN-93 re muh more thn the minimum requirement. In the present study, the requirement of vitmin B 1 of wening rts ws estimted y feeding nimls with vrious levels of vitmin B 1. We showed tht inresed energy expenditure deresed the onentrtion of ody vitmin B 1 in rts fed the minimum mount of vitmin B 1. MATERIALS AND METHODS Chemils. Thimin hydrohloride (C 12 H 17 ClN 4 OS- HCl, moleulr weight, ) ws purhsed from Wko Pure Chemil Industries, Ltd. (Osk, Jpn). All other hemils used were of the highest purity ville from ommeril soures. Animl experiments. The re nd tretment of experimentl nimls onformed to the guidelines for E-mil: kshit@sh.usp..jp the ethil tretment of lortory nimls set y the University of Shig Prefeture (Shig, Jpn). The room temperture ws mintined t ~2 C nd t 6% humidity. A 12-h light drk yle ws mintined. Body weight nd food intke were mesured every dy t 9 m. Experiment 1 (determintion of the minimum requirement of vitmin B 1 for mximum growth in wening rts). Thirty wening mle Wistr rts (ge, 3 wk old) were purhsed from CLEA Jpn, In. (Tokyo, Jpn). They were immeditely divided into six groups of five. They were fed diets ontining vrious mounts of thimin (Tle 1) for 25 d. Rts were kept in individul CL-31 Rt Metolism Cges (CLEA Jpn). On the lst dy of the experiment, 24-h urine smples (9 m to 9 m) were olleted in mer ottles ontining 1 ml of 1 mol/l HCl, nd were stored t 225 C. Rts were killed t ~9 m on dy 25 to mesure tissue weights. The ontents of vitmin B 1 in lood nd liver, nd urine were nlyzed. Experiment 2 (effet of swimming on the onentrtions of vitmin B 1 ). Twenty-six wening mle Wistr rts (ge, 3 wk old) were purhsed from CLEA Jpn. They were immeditely divided into two groups of 13. They were fed diets ontining the minimum mount of thimin (.786 mg thimin/kg diet) or suffiient mount of vitmin B 1 (4.72 mg thimin/kg diet) (Tle 1). Rts were kept in individul CL-31 Rt Metolism Cges (CLEA Jpn). To trin rts how to swim, ll 26 rts were fored to swim over period of 14 d. Then, eh group ws sudivided into two groups (swimming nd non-swimming) nd fed for nother 18 d. The four groups were set s follows: group 1 (nonswimming,.786 mg thimin/kg diet: n55); group 2 (swimming,.786 mg thimin/kg diet: n58); group 3 87

2 88 Shit K nd Fukuwtri T Tle 1. Compositions of the diets. mg.618 mg.786 mg 1.57 mg 2.36 mg 4.72 mg (mount sed on AIN-93-VX) Csein l-methionine Geltinized ornstrh Surose Corn oil Minerl mixture (AIN-93-M-MX) 1 Vitmin mixture (AIN-93-VX ontining holine itrtrte, vitmin B 1-free) 1 Thimin-HCl (mg/kg of diet) V.B 1: vitmin B 1. Eh vlue is expressed s g/kg of diet exept for vitmin B 1, whih is presented s mg/kg of diet. 1 Reeves (3). (non-swimming, 4.72 mg thimin/kg diet: n55); nd group 4 (swimming, 4.72 mg thimin/kg diet: n58). Rts in the swimming group were fored to swim until exhustion on lternte dys over 18 d (4, 5). In rief, swimming ws rried out in running-wter pool ( mm) filled to depth of 35 mm with wter mintined t ~35 C. The irulting wter urrent ws reted with pump. When rts were fored to swim, weight equl to 3% of the ody weight of the rt ws tied to the se of its til. They were judged to e exhusted if they ould not ome to the wter surfe to rethe in,7 s. On the lst dy of the experiment, 24-h urine smples (9 m to 9 m) were olleted in mer ottles ontining 1 ml of 1 mol/l HCl, nd were stored t 225 C. Rts were killed t ~9 m on dy 25 to mesure tissue weights nd the ontents of vitmin B 1 in lood, liver, nd urine. Biohemil nlyses. The ontents of thimin in the liver, lood, nd urine were nlyzed y the highperformne liquid hromtogrphy (HPLC) post-lel method using fluoresene detetion (6). Sttistil nlyses. Vlues re expressed s the mens6se. Sttistil signifine ws determined y one-wy ANOVA followed y Tukey s multiple omprison test (Fig. 1, Fig. 2, nd Tle 2). In the dt of Tle 3, Student s t-test ws performed to ompre the group of non-swimmers nd swimmers on the sme diet. Differenes with p,.5 were onsidered to e sttistilly signifint. Grph Pd Prism version 5. (Grph Pd Softwre, Sn Diego, CA, USA) ws used for ll nlyses. RESULTS Experiment 1 (determintion of the minimum requirement of vitmin B 1 for mximum growth in wening rts) Food intke nd gin in ody weight. The vitmin B 1 mount in AIN-93 vitmin mixture is 6. mg of thimin-hcl (4.72 mg of thimin) per kg of diet (3). We tenttively postulted tht this mount of thimin is the est for optimum growth of wening rts. It is thought tht the mount of vitmin B 1 in suh diet is severltimes higher thn the minimum mount required for the optimum growth of wening rts. We investigted the minimum requirement of vitmin B 1 for wening Wistr rts. Figure 1 shows the dily hnges in food intkes nd gin in ody weight of wening rts fed mg,.618 mg,.786 mg, 1.57 mg, 2.36 mg or 4.72 mg thimin per kg of diet for 25 d. The optimum growth ws hieved in the diet ontining.786 mg thimin/kg diet ut not the diet ontining.618 mg thimin/kg diet. From these results, we speulted tht the interim minimum requirement of dietry thimin to hieve optimum growth for wening rts ws.786 mg thimin/kg diet. Tissue weights. We mesured vrious tissue weights of rts fed the mg,.618 mg,.786 mg, 1.57 mg, 2.36 mg, or 4.72 mg thimin diet for 25 d. The results re shown in Tle 2. Only in the group fed the mg thimin diet were ll of the mesured tissue weights (exept for the rin), ody weight, nd food intke lower ompred with those for the other groups fed diets ontining thimin. These results suggest tht the minimum requirement of thimin for wening rts might e dietry onentrtion of round.618 mg thimin/kg diet. Conentrtions of vitmin B 1 in the rin, lood, liver, nd urine. We investigted the effet of dietry thimin ontent on the onentrtion of vitmin B 1 in the rin (Fig. 2A), liver (Fig. 2B), lood (Fig. 2C), nd urine (Fig. 2D). The onentrtions of vitmin B 1 in the rin, liver,

3 Exerise Inreses Consumption of Vitmin B 1 in Rts A 2 15 B Food intke (g/d) 1 Body weight gin (g) d d Fig. 1. Effet of dietry mount of vitmin B 1 on food intke (A) nd gin in ody weight (B) of wening Wistr rts (Experiment 1). Rts hd d-liitum ess to n ordinry purified diet ontining vrious mounts of vitmin B 1 for 25 d. The dietry vitmin ontents were:, mg thimin/kg diet;,.618 mg thimin/kg diet;,.786 mg thimin/kg diet;, 1.57 mg thimin/kg diet;, 2.36 mg thimin/kg diet;, 4.72 mg thimin/kg diet. Eh symol represents the men6se for 5 rts; the mens on the lst dy of the experiment with supersript letters without ommon letter differ, p,.5, determined y one-wy ANOVA followed y Tukey s multiple omprison test. Tle 2. Effet of dietry vitmin B 1 ontent on the tissue weights of rts (Experiment 1). mg.618 mg.786 mg 1.57 mg 2.36 mg 4.72 mg (mount sed on AIN-93-VX) Body weight Brin Lung Liver Kidney Hert Spleen Testis V.B 1: vitmin B 1. Mle wening rts were fed the diet desried for 25 d. Vlues re mens6se for 5 rts; the mens in row with supersript letters without ommon letter differ, p,.5, determined y one-wy ANOVA followed y Tukey s multiple omprison test. nd lood were lower in the group fed the mg thimin diet thn those in the thimin-ontining diets. There were no differenes in the onentrtion of vitmin B 1 in the rin mong rts fed diets ontining.618 mg,.786 mg, 1.57 mg, 2.36 mg, nd 4.72 mg thimin/kg diet. These findings suggest tht the diet ontining.618 mg thimin/kg diet ould mintin the optimum onentrtion of vitmin B 1 in the rin. The onentrtions of vitmin B 1 in the liver nd lood inresed linerly in ordne with inresing dietry mounts of vitmin B 1 up to 1.57 mg/kg diet nd therefter steeply inresed up to 2.36 mg thimin/kg diet. There were no signifint differenes in the onentrtions of vitmin B 1 in the liver or lood etween the groups fed diets ontining 2.36 mg nd 4.72 mg thimin/kg diet. These findings suggest tht the diet ontining 2.36 mg thimin/kg diet ould mintin the optimum onentrtion of vitmin B 1 in

4 9 Shit K nd Fukuwtri T Brin V.B 1 (nmol/g) A Dietry V.B 1 (mg/kg diet) Liver V.B1 (nmol/g) B, Dietry V.B 1 (mg/kg diet) Blood V. B1 (nmol/ml) 4 C Dietry V.B 1 (mg/kg diet) Urine V. B1 (nmol/dy) 8 D Dietry V.B 1 (mg/kg diet) Fig. 2. Effet of dietry mounts of vitmin B 1 on vitmin-b 1 onentrtions in the rin (A), liver (B), lood (C), nd urine (D) of wening Wistr rts (Experiment 1). Rts hd d-liitum ess to n ordinry purified diet ontining vrious mount of vitmin B 1 for 25 d. Eh point represents the men6se for 5 rts; the mens with supersript letters without ommon letter differ, p,.5, determined y one-wy ANOVA followed y Tukey s multiple omprison test. Tle 3. Effets of swimming on gin in ody weight, food intke, liver weight nd onentrtions of vitmin B 1 in the liver, lood, nd urine when rts were fed diet ontining suffiient or minimum requirement of vitmin B 1 (Experiment 2). Suffiient mount of V.B mg/kg diet (mount sed on AIN-93-VX) Minimum requirement of V.B mg/kg diet Non-swimming Swimming Non-swimming Swimming Body weight gin (g/18 d) * Food intke (g/18 d) Liver weight (g) V.B 1 Liver (nmol/g) * Blood (pmol/ml) * Urine (nmol/) * V.B 1: vitmin B 1, BW: ody weight. Vlues re mens6se for 5 nd 8 rts in the groups of non-swimming nd swimming, respetively. * Signifint differene t p,.5 ompred to non-swimming nd swimming rts eting the sme diet s lulted y Student s t-test. the liver nd lood. The rek point on 1.57 mg thimin/kg diet is interesting, ut we do not know wht it mens. The urinry exretions of vitmin B 1 inresed in ordne with inresing dietry ontent of thimin (Fig. 2D). The urinry exretion of vitmin B 1 ws not deteted in rts fed the diet ontining.618 mg/ kg diet, while it ws distintly oserved in rts fed the diet ontining.786 mg/kg diet. However, signifint inrese in urinry exretion of vitmin B 1 ws not oserved etween the groups fed diets ontining.618 mg nd.786 mg/kg diet. A signifint inrese in urinry exretion of vitmin B 1 ws oserved etween the groups fed diets ontining 2.36 mg nd 4.72 mg/kg diet. Experiment 2 (effet of swimming on the onentrtions of vitmin B 1 in the liver, lood, nd urine) Swimming on lternte dys for 18 d did not ffet ody weight gin, food intke, liver weight, onentrtions of vitmin B 1 in the liver nd lood, or urinry

5 Exerise Inreses Consumption of Vitmin B 1 in Rts 91 exretion of vitmin B 1 in rts fed diet ontining suffiient mount of thimin (Tle 3). Conversely, in rts fed diet low in thimin, ody weight gin, onentrtions of vitmin B 1 in the liver nd lood, nd urinry exretion of vitmin B 1 were deresed y swimming. DISCUSSION The requirement for vitmin B 1 in humns is sed on energy expenditure. For exmple, the estimted verge requirement of vitmin B 1 (thimin) for Jpnese sujet is.35 mg/1, kl energy expenditure (7). This onsidertion is sed on the ft tht 2-oxoid dehydrogenses (whih re involved in the retions leding to the formtion of denosine triphosphte (ATP)) need thimin diphosphte s oenzyme s well s niotinmide denine dinuleotide (NAD) nd oenzyme A (CoA). Shit et l. (4) lrified tht, in mie, lood levels of NAD re deresed y severe exerise nd tht dministrtion of niotinmide ( preursor of NAD) improves their endurne. However, we ould not find ny reports showing inresed energy expenditure deresed the onentrtion of vitmin B 1 in tissues nd lood or urinry exretion of vitmin B 1. This disrepny with our result might e used y the mount of vitmin B 1 in the diet, sine most of the experiments were rried out with diet ontining suffiient mount of vitmin B 1. The minimum requirement of vitmin B 1 for rts hs een reported (8). The dt suggest tht the thimin requirement for optimum growth of wening rts fed hemilly defined diet is ~.55 mg thimin/kg diet (8). This vlue is sustntilly elow the AIN-93 reommendtion of 6. mg thimin hydrohloride/kg diet (4.72 mg thimin/kg diet) (3). Merer et l. (9) reported tht thimin levels less thn 1.25 mg thimin/ kg diet were too low for mle rts to reh optimum growth. The differene etween the two experiments (8, 9) might e relted to differenes in dietry ingredients (e.g., soures of proteins nd rohydrtes). Thus, we investigted the minimum requirement of vitmin B 1 in diet sed on AIN-93 (3) nd speulted the minimum vitmin B 1 requirement tht n sustin ody weight (3). A diet ontining.618 mg thimin/kg diet ould not sustin the sme level of growth otined in the diet ontining 4.72 mg thimin/kg diet (Fig. 1), while diet ontining more thn.786 mg thimin/kg diet ould mintin mximl growth. Thus, we speulted tht the interim minimum requirement of dietry thimin to hieve optimum growth for rts ws.786 mg thimin/kg diet. To onfirm tht the vlue is round the minimum requirement of vitmin B 1 for growing wening rts, severl tissue weights (rin, lung, liver, kidney, hert, spleen, nd testis) were ompred with those in rts fed diets ontining higher mounts of thimin (Tle 2). As result, none of the tissue weights mong the groups fed diets ontining more thn.618 mg thimin/kg diet different (Tle 2). The tissue weights of the rts fed the diet ontining.618 mg thimin/kg of diet showed tht level ould sustin norml weights. The onentrtion of vitmin B 1 in tissue yielded less definitive results (Tle 2). The onentrtion of vitmin B 1 in the rin ould e sustined t the sme level in the group fed diet ontining 4.72 mg thimin/ kg diet s in the group fed diet ontining.618 mg thimin/kg diet. The hnges in the onentrtions of vitmin B 1 in the liver nd lood re interesting (Fig. 2B nd C): these inresed linerly following inresing dietry mounts of vitmin B 1 up to 1.57 mg/kg diet, nd steeply inresed up to 2.36 mg thimin/kg diet, ut showed no signifint differenes etween 2.36 mg nd 4.72 mg thimin/kg diet. These results suggest tht the diet ontining 2.36 mg thimin/kg diet ould mintin the optimum onentrtion of vitmin B 1 in the liver nd lood. The results lso suggest tht the requirements of vitmin B 1 in the rin nd liver might not e the sme. A signifint inrese in urinry exretion of vitmin B 1 ws oserved in the groups fed diets ontining.618 mg ompred with.786 mg thimin/kg diet nd in the group with 2.36 mg ompred with 4.72 mg thimin/kg diet (Fig. 2D). In other words, two rek points were oserved. Generlly speking, urine vitmin exretion represents surplus intke of vitmin (1 12). The seond rek point is onsidered to indite tissue sturtion point euse the onentrtions in liver nd lood were lmost the sme for the two groups of 2.36 mg nd 4.72 mg/kg diet (Fig. 2B nd C). In other words, rts nnot retin nymore thimin in the ody even when they re fed diet ontining more thn 2.36 mg thimin/kg diet. The extr mount of thimin is eliminted vi urine. The mening of the first rek point is osure, ut we suggest tht the point might indite minimum requirement of vitmin B 1. From these results nd onsidertion, we speulted tht the interim minimum requirement of dietry thimin to hieve optimum growth for wening rts ws lose to or slightly ove.618 mg thimin/kg diet. The thimin requirement of rts is ontroversil sujet. However, we speulted tht requirement for wening mle Wistr rts (ge, 3 wk old; men ody weight, 35 g) is round.786 mg/kg diet. This vlue is severl-fold lower thn tht of the AIN-93 diet (4.72 mg of thimin/kg diet) (3) nd Ntionl Reserh Counil guideline (3.1 mg of thimin/kg diet) (13). Exerise n inrese the requirement for vitmin B 1. Some uthors elieve tht it does (14, 15), wheres other uthors do not (16 18). To sertin if exerise inreses the requirement of vitmin B 1, we investigted whether the onentrtions of vitmin B 1 in the liver nd lood were redued when rts were fored to exerise on lternte dys fter eing fed diet ontining suffiient mount of thimin (4.72 mg thimin/ kg diet) or diet ontining minimum requirement of thimin (.786 mg thimin/kg diet). In the present study, we demonstrted tht requirement of vitmin B 1 inresed following inreses in energy expenditure. But, if the vitmin B 1 intke is suffiient, even if the liver

6 92 Shit K nd Fukuwtri T nd lood vitmin B 1 onentrtions re redued y inresed energy expenditure with exerise, the mount of derese is too smll to mesure. As result, we demonstrted the redution of vitmin B 1 levels in the ody following exerise, suggesting inreses in thimin onsumption within the ody following energy expenditure. Aknowledgments This investigtion is prt of results in Studies on onstrution of evidene to revise the dietry referene intkes for Jpnese: eluidtion of lne of mironutrient nd lrge mount of nutrient intke (prinipl investigtor, Ktsumi Shit), whih ws supported y Reserh Grnt for Comprehensive Reserh on Crdiovsulr nd Life-Style Relted Diseses from the Ministry of Helth, Lor nd Welfre of Jpn. The uthors delre tht they hve no ompeting interest. REFERENCES 1) Ier FL, Blss JP, Brin M, Leevy CM Thimin in the elderly-reltion to loholism nd to neurologil degenertion disese. Am J Clin Nutr 36: ) Vn Dm B Vitmins nd sports. Br J Sports Med 12: ) Reeves PG Components of the AIN-93 diets s improvements in the AIN-76A diet. J Nutr 127: 838S 841S. 4) Shit K, Mtsumoto K, Fushiki T, Sugimoto E Effets of exerise on the metolism of NAD in rts. Biosi Biotehnol Biohem 58: ) Mtsumoto K, Ishihr K, Tnk K, Inoue K, Fushiki T An djustle-urrent swimming pool for the evlution of endurne pity of mie. J Appl Physiol 81: ) Fukuwtri T, Suzuur C, Sski R, Shit K. 24. Ation site of isphenol A s metoli disruptor lies in the tryptophn-niotinmide onversion pthwy. Shokuhin Eiseigku Zsshi 45: (in Jpnese). 7) Ministry of Helth, Lour, nd Welfre of Jpn. 29. Dietry Referene Intkes for Jpnese (21). Ministry of Helth, Lour, nd Welfre, Tokyo. 8) Rins TM, Emmert JL, Bker DH, Shy NF Minimum thimin requirement of wening Sprgue-Dwley outred rts. J Nutr 127: ) Merer LP, Dodds SJ, Gustfson JM The determintion of nutritionl requirements: modeling pproh. Nutr Rep Int 34: ) Fukuwtri T, Shit K. 28. Urinry wter-solule vitmin nd their metolites ontents s nutritionl mrkers for evluting vitmin intkes in young Jpnese women. J Nutr Si Vitminol 54: ) Fukuwtri T, Yoshid E, Tkhshi K, Shit K. 21. Effet of fsting on the urinry exretion of wter-solule vitmins in humns nd rts. J Nutr Si Vitminol 56: ) Imi E, Tsuji T, Sno M, Fukuwtri T, Shit K Assoition etween 24 hour urinry -toopherol tolite, 2,5,7,8-tetrmethyl-2(2 -roxyethyl)-6-hydroxyhromn (-CEHC) nd -toopherol intke in intervention nd ross-setionl studies. Asi P J Clin Nutr 2: ) Ntionl Reserh Counil Nutrient Requirements of Lortory Animls, 4th ed. Ntionl Ademies Press, Wshington, DC. 14) Vn der Beek EJ, vn Dokkum W, Shrijver J, Wedel M, Gillrd AWK, Wesstr A, vn de Weerd H, Hermus RJJ Thimin, rioflvin, nd vitmins B-6 nd C: impt of omined restrited intke on funtionl performne in mn. Am J Clin Nutr 48: ) StoA, Shimoym Y, Ishikw T, Murym N Dietry thimin nd rioflvin intke nd lood thimin nd rioflvin onentrtions in ollege swimmers undergoing intensive trining. Int J Sport Nutr Exer Met 21: ) Fogelholm GM, Himerg JJ, Alopeus K, Gref CG, Lkso JT, Lehto JJ, Musslo-Ruhm H Dietry nd iohemil indies of nutritionl sttus in mle thletes nd ontrols. J Am Coll Nutr 11: ) Vn der Beek EJ, vn Dokkum W, Wedel M, Shrijver J, vn den Berg H Thimin, rioflvin nd vitmin B6: impt of restrited intke on physil performne in mn. J Am Coll Nutr 13: ) Frnk T, Kühl M, Mkowski B, Bitsh R, Jhreis G, Hüsher J. 2. Does 1-km wlking ffet inditors of vitmin sttus? Int J Vitm Nutr Res 7: