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1 Cratin Rtntion Following 3 Formulations of Cratin Ingstion 37 JEPonlin Journal of Exris Physiologyonlin Offiial Journal of Th Amrian Soity of Exris Physiologists (ASEP) ISSN An Intrnational Eltroni Journal Volum 6 Numr 2 May 2003 Nutrition an Exris DIFFERENCES IN CREATINE RETENTION AMONG THREE NUTRITIONAL FORMULATIONS OF ORAL CREATINE SUPPLEMENTS MIKE GREENWOOD, RICHARD KREIDER, CONRAD EARNEST, CHRISTOPHER RASMUSSEN, ANTHONY ALMADA Sport & Exris Nutrition Laoratory, Cntr for Exris, Nutrition & Prvntiv Halth Rsarh Dpartmnt of Halth, Human Prforman & Rration, Baylor Univrsity, Wao, Txas ABSTRACT DIFFERENCES IN CREATINE RETENTION AMONG THREE NUTRITIONAL FORMULATIONS OF ORAL CREATINE SUPPLEMENTS. Mik Grnwoo, Rihar Krir, Conra Earnst, Christophr Rasmussn, Anthony Almaa. JEPonlin. 2003;6(2): Prvious rsarh has iniat that ratin rtntion is influn y intramusular ratin onntration an xtrallular onntrations of gluos an insulin. This stuy xamin whthr iffrnt nutritional stratgis afft whol oy ratin rtntion. Spifially, 16 mals with no history of ratin supplmntation partiipat in this stuy. Sujts onat 24-hr urin sampls for 4 ays. Aftr an initial ontrol ay, sujts wr math aoring to oy mass an assign to ingst in a singl lin mannr ithr 5 g of xtros (D), 5 g of ratin monohyrat (CM), 5 g of CM + 18 g xtros (C+D), or an ffrvsnt ratin (EC) supplmnt (5 g of ratin + 18 g xtros mg of soium [as soium aronat an iaronat] mg of potassium [as potassium iaronat]) four tims/ay for 3 ays. Cratin rtntion was stimat y sutrating total urinary ratin xrtion from total supplmntal ratin intak ovr th 3 ay prio. Data wr analyz y ANOVA. Rsults rval that ratin rtntion was inras following ratin supplmntation in all groups (D=0±0; CM= 36.6±9; C+D=48.0±7; EC=37.8±8 g, p=0.001). Howvr, ratin rtntion in th C+D group was signifiantly gratr than th CM group whil no iffrns wr osrv twn th EC an CM groups. This rsult in a gratr prntag of ratin rtntion in th CD group (D= 0±0; CM=61±15; C+D=80±11; EC=63±13 %, p=0.001). Ths prliminary finings suggst that in aoran with prvious rsarh, ingsting xtros (18 g) with CM (5 g) augmnts whol oy ratin rtntion whil EC supplmntation appars to no mor fftiv than ingsting CM alon. Ky Wors: Exris, Sport Nutrition, Ditary Supplmntation, Ergogni Ai INTRODUCTION

2 Cratin Rtntion Following 3 Formulations of Cratin Ingstion 38 Cratin supplmntation (5 g takn 4 tims/ay) has n rport to inras musl ratin an phosphoratin ontnt y 5 to 30%. Howvr, a signifiant amount of intra-sujt variaility has n rport in th litratur rgaring th magnitu that ratin stors ar inras in rspons to ratin loaing an how lvations in musl ratin ontnt afft prforman (1). Rsarh on th variaility in ratin rtntion has iniat that ratin uptak into th musl is influn y th amount of ratin in th musl for supplmntation, as wll as gluos-stimulat inras insulin rlas (2,3). In this rgar, stuis hav suggst that o-ingstion of ratin with larg amounts of gluos (97 g) an/or ominations of gluos an protin may nhan ratin storag (2-5). Consquntly, it has n propos that ratin storag may gluos an/or insulin pnnt (6). Thortially, o-ingstion of ratin with othr nutrints that hav n rport to afft insulin snsitivity an/or gluos availaility may nhan ratin rtntion (7). Ovr th last fw yars, a numr of ratin ontaining prouts hav n markt with laims to nhan ratin transport into musl. Most of ths ontain gluos with othr nutrints sign to optimiz ll volum an/or transport ratin or gluos (.g., taurin, glutamin, t). Aitionally, svral iffrnt forms of ratin hav n markt (liqui, any, gum, ffrvsnt, ratin itrat, t). For xampl, ffrvsnt ratin itrat prouts hav n markt as a mor optimal mans of ingsting ratin aus thy thortially nhan th suspnsion an soluility of th ratin in liqui, optimiz ph lvls to prvnt graation of ratin to ratinin, an ru purport gastrointstinal prolms that may intrfr with ratin transport in th gut. Although thr is som vin that ingsting ratin with larg amounts of gluos or gluos/protin optimizs ratin storag, littl is known whthr any othr typs of prouts promot ratin rtntion. Thrfor, th purpos of this pilot stuy was to xamin th ffts of ingsting svral nutritional stratgis sign to nhan ratin uptak on whol oy ratin rtntion. METHODS Sujts Sixtn apparntly halthy mals with no history of ratin us partiipat in this pilot stuy. All sujts in this invstigation partiipat in a familiarization sssion. During th familiarization sssion, sujts wr inform as to th xprimntal prours, omplt a prsonal/mial history form, xris history form, ratin supplmntation history form, an sign inform onsnt statmnts in ahrn with th human sujt s guilins of Arkansas Stat Univrsity an th Amrian Collg of Sports Miin. Sujt s sriptiv haratristis wr (man ±SD) 22.3±1.4 yrs, 82±8 kg, an 182±6 m. No sujt in this trial was a vgtarian an all sujts rportly onsum aily its inlusiv of mat. Supplmntation Protool Sujts onat a 24-hr urin sampl on th ay pring th initiation of supplmntation in orr to stalish th sujt s normal aily xrtion of ratin in rspons to thir normal it. Aftr this ontrol ay, sujts wr math aoring to total oy mass an ranomly assign to ingst in a singl-lin mannr on of th following supplmnts four tims aily for 3-. Plao (P): 5 g of xtros with on 0.5 g apsul of orn starh. Cratin Monohyrat (CM): 5 g of CM with on 0.5 g apsul of orn starh. Cratin Monohyrat + Dxtros (CM + D): 5 g of CM + 18 g xtros; Effrvsnt Cratin (EC) 5 g of ratin itrat + 18 g xtros mg of soium [as soium aronat an iaronat] mg of potassium [as potassium iaronat]) Sujts wr instrut to mix th powr supplmnts with watr an to ingst th supplmnts at 8:00 a.m., 12:00 p.m., 4.00 p.m., an 8.00 p.m. ah ay in orr to stanariz supplmnt intak. Dxtros an ratin powrs wr pla in gnri singl srving pakts for singl-lin aministration an wr ompris of similar msh siz, txtur, tast, an apparan. Th ratin monohyrat us in th stuy was from SKW (Trotsrg, Grmany) an th ffrvsnt ratin was otain from FSI Nutrition (Boys Town, Nraska). Sujt

3 Cratin Rtntion Following 3 Formulations of Cratin Ingstion 39 omplian in taking th supplmnts was vrifi aily y rsarh assistants an all sujts wr instrut to maintain thir rgular ating haits uring th invstigation prio. Sujts itary intak was monitor with aily nutritional logs that wr turn in ah morning an it was not that all sujts wr mat atrs. Prours During th familiar sssion, sujts wr instrut y th primary invstigator on how to ror nutritional intak on th provi nutritional log shts. In aition, th primary invstigator issminat in a singl lin mannr th rsptiv ratin prouts along with a vral an writtn sription of th supplmntation protool. Sujts wr provi ight 3 L urin olltion ontainrs in orr to ollt 24 hr urin sampls ovr th ours of th stuy an wr also rqust to ror th numr of tims thy urinat ah ay. Th 24 hr aslin urin sampl tim paramtr was initiat at 8 am th ay for supplmntation protools gan. Sujts wr ask to rfrigrat thir urin sampls uring th 24 hr tim prio. Sujts rport aily to th Human Prforman Laoratory twn 7 an 8 am in orr to rop off urin sampls. Sujts also turn in aily nutritional intak logs, whih inlu typ an amount of flui ingst ovr th 24 hr tim prio. Urin volum an flui intak for th 24 hr prio wr ror. Urin sampls wr vortx an a stanar qualitativ urinalysis was prform to assss urin spifi gravity (Chm Strip 10SG, Roh Diagnostis, Inianapolis, IN). In aition, approximatly 10 ml of urin was transfrr into lal urin storag tus an stor at -80 C. Urin sampls wr shipp on ry i to rsarhrs in th Dpartmnt of Biomial Sins, Qun s Mial Cntr, at th Univrsity of Nottingham, Englan for lin analysis of ratin an ratinin lvls using stanar high prforman liqui hromatography (HPLC) mthos (2,3,5). Daily ratin an ratinin xrtion (g) wr trmin y multiplying aily xrtion (g/l) y urin volum xprss in L. Daily ratin rtntion was alulat y sutrating aily ratin xrtion (g) from aily supplmntal ratin (20 g). Cumulativ ratin rtntion was trmin y sutrating th total amount of ratin xrt ovr th 3- supplmntation prio from th total amount of ratin supplmnt to th it uring th 3-ay loaing prio (i.., 60 g). Prnt ratin rtntion was trmin y iviing th umulativ amount of ratin rtain ovr th supplmntation prio y th total amount of ratin supplmnt to th it. Statistial Analyss Data wr analyz y rpat masur ANOVA with LSD post-ho prours for all aily masurmnts. A fatorial ANOVA with LSD post-ho prour was us to assss all umulativ (i.., 3 ay) ata masurs. Data wr analyz using th SPSS for Winows vrsion statistial pakag (SPSS In., Chiago, IL). Statistial signifian was trmin as p<0.05. Data ar prsnt as mans±sd. RESULTS No signifiant intrations (p>0.05) wr osrv among groups in flui intak, urin spifi gravity, or urinary ratinin xrtion. Tal 1 prsnts man aily urin volum, ratin xrtion, an ratin rtntion osrv for th plao (P), ratin monohyrat (CM), ratin monohyrat xtros (C+D), an ffrvsnt ratin (EC) groups. No signifiant intrations wr osrv among groups in urin volum. Daily ratin xrtion xprss in g/l inras in all groups ingsting ratin uring th supplmntation prio in omparison to thir ontrol ay an th plao group. Signifiant iffrns wr also osrv among th ratin supplmntation tratmnts. Post-ho analysis rval that ratin xrtion was gratr in th CM an EC groups in omparison to th C + D group. Signifiant group ffts (p=0.001) wr also osrv among aily stimat ratin rtntions uring th 3- ratin-loaing prio. Avrag aily ratin rtntion was 0±0, 12.2±1.3, 16.1±2.2, an 12.6±2.5, g/ for th P, CM, C+D, an HP groups rsptivly. Post-ho analysis rval that avrag aily ratin rtntion was signifiantly gratr in th C+D group in omparison to th P, CM, an EC groups. This rsult in a gratr prntag of ratin rtntion in th CD group (D=0±0; CM=61±15; C+D=80±11; EC=63±13 %, p=0.001).

4 Cratin Rtntion Following 3 Formulations of Cratin Ingstion 40 Tal 1. Daily urin volum, urinary ratin xrtion, an stimat ratin rtntion osrv for th plao (P), ratin monohyrat (CM), ratin + xtros (C+D), an ffrvsnt ratin (EC) groups. Control Day 1 Day 2 Day 3 Urin Volum (L) P 1.50± ± ± ±0.45 CM 2.16± ± ± ±1.13 C+D 2.50± ± ± ±0.52 EC 1.73± ± ± ±1.70 Urin Cratin (g/l) P 0.14± ± ± ±0.06 CM 0.54± ±2.55 a 8.58±3.78 a 9.28±6.3 a C+D 0.30± ±1.54 a 3.00 ±1.40 a 6.42±3.72 a EC 0.28± ±2.10 a 8.01±3.00 a 7.00±6.42 a Cratin Rtntion (g/) P 0±0 0±0 0±0 CM 14.46± ± ±6.30 C+D 17.40± ± ± EC 12.72± ± ±6.30 a = p<0.05 iffrn from ontrol ay; = p<0.05 from th P group. = p<0.05 from th CM group; = p<0.05 from th C + D group. = p<0.05 from th EC group Figur 1 prsnts th stimat umulativ ratin rtntion xprss in grams osrv uring th 3 ay loaing prio. ANOVA rval signifiant iffrns among groups (p=0.001) in total ratin rtntion. Post-ho analysis iniat that ratin supplmntation inras whol oy ratin rtntion in all groups in omparison to P group. Howvr, ratin rtntion in th C+D group was signifiantly gratr (p<0.001) than th CM group whil no iffrns wr osrv twn th EC an CM groups. Figur 2 prsnts th stimat umulativ prntag of supplmntal ratin rtain uring th 3- loaing prio for th P, CM, C+D, EC groups, rsptivly. Furthr, signifiant iffrns (p=0.001) wr similarly osrv among groups whn ratin rtntion was xprss as a prntag of total ratin supplmnt in th it. Grams Plao Cratin Monohyrat Cratin + Dxtros Effrvsnt Cratin Figur 1. Thr-ay umulativ ratin rtntion for th plao (P), ratin monohyrat (CM), ratin + xtros (C + D), an ffrvsnt ratin (EC) groups. Data ar mans±sd. a=p<0.05 from plao. =p<0.05 from CM. =p<0.05 from C + D. =p<0.05 from EC. DISCUSSION Th major fining from this stuy is that ratin rtntion in th C (5g)+D (18 g) group was signifiantly gratr than th CM group an that EC+D supplmntation i not promot gratr ratin rtntion ompar to CM supplmntation. Ths finings ar important aus until now th only known mthos for nhaning ratin

5 Cratin Rtntion Following 3 Formulations of Cratin Ingstion 41 uptak hav n y o-ingstion of ratin with larg amounts of gluos (.g., g) an/or gluos an protin (~50 g ah) (2-5) or y ingsting low osags of D-Pinitol (7). Harris an oworkrs (1) wr among th first to show that th oral ratin monohyrat supplmntation (.g., 5 g, 4-6 tims pr ay, for 2 or mor ays) signifiantly inras total ratin ontnt of th quarips fmoris musl. It was furthr osrv that th gratst uptak y skltal musl ourr in sujts with a low initial total ratin ontnt (1). Svral yars latr, Grn an ollagus (2,3) monstrat via analysis of musl iopsy, urin, an plasma sampls that ingsting 5 g of ratin monohyrat, follow 30-minuts latr y ingsting 93 g of simpl arohyrat in solution four tims ah ay for 5 ays rsult in an inras in musl phosphoratin, ratin, an total ratin ompar to ratin Prnt % Plao Cratin Monohyrat Cratin + Dxtros Effrvsnt Cratin Figur 2. Prntag of ratin rtain uring th 3 ay loaing prio for th plao (P), ratin monohyrat (CM), ratin + xtros(c + D) an ffrvsnt ratin (EC) groups. Data ar mans±sd. a=p<0.05 from plao. =p<0.05 from CM. =p<0.05 from C + D. =p<0.05 from EC. ingstion alon. Ths rsarhrs also foun that ratin plus arohyrat ingstion ramatially lvat insulin onntrations an glyogn synthsis. Ths finings l to th prmis that ratin aumulation uring ratin supplmntation in humans appars to miat in part y insulin. Invstigation into this phnomnon has shown that ingsting 35 g of arohyrat with ah os of ratin may promot gratr training aaptations than ingsting ratin alon (4) an that th omination of arohyrat (47g, 50g, 97g) an protin (50g) will also augmnt ratin rtntion (5). Though this phnomnon is intrsting, it an onrous to th athlt, as on woul hav to onsum an xtra 560-1,500 Kals with ratin in orr to promot ths aaptations. In a ompanion stuy to th prsnt invstigation, w valuat whthr D-pinitol supplmntation uring ratin loaing woul afft whol oy ratin rtntion in mal sujts (7). Sin D-pinitol has n rport to possss insulin-lik proprtis (8,9) an stimulat gluos uptak (10,11) it was thoriz that th omination of ratin monohyrat an D-pinitol might inras ratin rtntion. W foun that o-aministration of ratin monohyrat (5g) with low-oss of D-pinitol (0.5g, twi/ay) offr a non-alori mans of augmnting whol oy ratin stors. Howvr, sin D-pinitol is fairly xpnsiv, it has yt to havily markt for onsumr us in rlation to augmnting ratin rtntion. Consquntly, thr has n intrst in trmining whthr othr nutritional intrvntions may augmnt ratin rtntion suh as th prsnt stuy suggsting lowr osags (18 g) of arohyrat supplmntation that ar mor afforal. Anothr intrsting fining in this stuy was that ffrvsnt ratin supplmntation i not promot gratr whol oy ratin rtntion ompar to ratin monohyrat supplmntation alon. Th primary iffrn twn ths two stratgis is that ffrvsnt ratin provis ratin itrat rathr than ratin monohyrat in a arohyrat ontaining ffrvsnt rink thortially sign to optimiz ratin livry to th musl. This fining ontrasts markt laims that ffrvsnt ratin is a ttr mans of promoting whol oy ratin rtntion than ratin monohyrat. Furthr, that improving th mixing haratristis of ratin in flui through aing ffrvsn; optimizing th ph of th flui ratin is mix to prvnt graation to ratinin; an/or attmpting to minimiz GI istrss affts whol oy ratin rtntion. Although on stuy has rport rgogni nfit from ffrvsnt ratin itrat supplmntation (12), w know of no othr invstigations that hav

6 Cratin Rtntion Following 3 Formulations of Cratin Ingstion 42 xamin th ffiay of ffrvsnt ratin itrat on whol oy ratin rtntion. Howvr, prsnt finings suggst that ffrvsnt ratin may atually a lss ffiint mans of augmnting whol oy ratin stors. In this rgar, th prsnt stuy rval that aing 18 g of xtros to ratin monohyrat promot gratr whol oy ratin rtntion than ingsting ratin monohyrat alon or ffrvsnt ratin. Sin th ffrvsnt ratin also ontain 18 g of xtros, on woul xpt that ffrvsnt ratin woul at last promot a similar inras in whol oy ratin rtntion than th ratin + xtros group. Sin th ffrvsnt ratin group promot similar whol oy ratin rtntion than ratin monohyrat alon, it oul argu that ratin itrat is a lss ffiint form of ratin than ratin monohyrat. Spulativly, this ru asorption ffiiny may u to variations in intstinal an/or musl asorption haratristis of ratin itrat in omparison to ratin monohyrat. Howvr, mor rsarh is n to xamin possil iffrns twn ratin itrat an ratin monohyrat for onlusions an rawn. In summary, rsults of this pilot stuy iniat that ingsting xtros (18 g) with CM (5 g) signifiantly augmnts whol oy ratin rtntion ovr a thr-ay prio. This fining is important aus to at, prvious invstigations hav utiliz largr quantitis of arohyrat (35-97g) to nhan ratin rtntion. Thrfor, as on th finings of this invstigation, ratin rtntion an inras vn with rlativly small amounts of simultanous arohyrat ingstion. Furthr, ffrvsnt ratin has n markt as a highly fftiv mtho to nhan ratin uptak ut th rsults of this pilot stuy iniat that ratin itrat (EC) supplmntation is no mor fftiv than ingsting CM alon. Whil th rsults of this stuy support prvious rsarh, aitional rsarh is warrant to xamin th possil influn that varying osags of ratin monohyrat an xtros supplmntation may hav on lvls of whol oy ratin rtntion. Furthr, it is vital to ontinu th lin of rsarh rgaring th safty an ffiay of th svral iffrnt forms of ratin that ar ing markt toay (liqui, any, gum, ffrvsnt, ratin itrat, t). ACKNOWLEDGMENTS W woul lik to thank th sujts who partiipat in this stuy an th laoratory assistants in th Human Prforman Laoratory at Arkansas Stat Univrsity who assist in ata aquisition an analysis. This stuy was fun in part y MtaRspons Sins (Laguna Nigul, CA). Invstigators from Arkansas Stat ollt, analyz an intrprt ata from this stuy an hav no finanial intrst in th outom of rsults rport. Prsntation of rsults in this stuy os not onstitut norsmnt y th rsarhrs or th institutions that thy ar affiliat of th nutrints invstigat. Currnt arss for M. Grnwoo, PhD, CSCS*D, R.B. Krir, PhD, EPC an C. Rasmussn, MS, CSCS, EPC is Th Exris & Sport Nutrition Laoratory, Dpartmnt of Halth, Human Prforman & Rration Cntr for Exris, Nutrition, Prvntiv Halth, Rsarh, Baylor Univrsity, P.O. Box Wao, TX Currnt arss for C. P. Earnst, PhD is Th Coopr Institut, Division of Epimiology & Clinial Appliations, Prston Roa, Dallas TX Currnt arss for A.L. Almaa, MS is MtaRspons Sins, In., Town Cntr Driv, # 211, Laguna Nigul, CA Arss orrsponn to: Mik Grnwoo, PhD, CSCS*D. Arss for Corrsponn: Mihal Grnwoo, PhD, CSCS *D, Exris & Sport Nutrition Laoratory, Dpartmnt of HHPR, Baylor Univrsity, PO Box 97313, Wao, TX Phon: (254) ; FAX: (254) ; [email protected]

7 Cratin Rtntion Following 3 Formulations of Cratin Ingstion 43 REFERENCES 1. Harris RC, Sorlun K, Hultman E. Elvation of ratin in rsting an xris musl of normal sujts y ratin supplmntation. Clin Si (Colh) 1992;83: Grn AL, Hultman E, Maonal IA, Swll DA an Grnhaff PL. Carohyrat ingstion augmnts skltal musl ratin aumulation uring ratin supplmntation in humans. Am J Physiol 1996;271:E Grn AL, Simpson EJ, Littlwoo JJ, Maonal IA an Grnhaff PL. Carohyrat ingstion augmnts ratin rtntion uring ratin fing in humans. Ata Physiol San 1996;158: Stout J, Ekrson J, Noonan D, Moor G an Culln D. Effts of 8 wks of ratin supplmntation on xris prforman an fat-fr wight in footall playrs uring training. Nutrition Rsarh 1999;19: Stng GR, Simpson EJ an Grnhaff PL. Protin- an arohyrat-inu augmntation of whol oy ratin rtntion in humans. J Appl Physiol 2000;89: Hauglan RB an Chang DT. Insulin fft on ratin transport in skltal musl (38464). Pro So Exp Biol M 1975;148:1-4, Grnwoo M, Krir R, Rasmussn C, Almaa A an Earnst CP. Analysis of various nutritional formulation stratgis on whol oy ratin rtntion. JEPonlin 2001;4: Holman GD an Kasuga M. From rptor to transportr: insulin signaling to gluos transport. Diatologia 1997;40: Whit MF. Th insulin signaling systm an th IRS protins. Diatologia 1997;40 Suppl 2: S Bats SH, Jons RB an Baily CJ. Insulin-lik fft of pinitol. Br J Pharmaol 2000;130: Narayanan CR, Joshi DD, Mujummr AM an Dhnk VV. Pinitol, a nw anti-iati ompoun from th lavs of Bougainvilla sptailis. Curr Si 1987;56: Stout J, Ekrson J, Ersol K, Moor G, Prry S, Housh T, Bull A, Cramr J, Bathja A. Efft of ratin loaing on nuromusular fatigu thrshol. J Appl Physiol 2000;88: