British Journal of Nutrition

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1 (21), pge 1 of 5 q The Authors 21 doi:1.117/s Aute supplementtion with keto nlogues nd mino ids in rts during resistne exerise Rosemeire Dnts de Almeid 1,2,3, Edurdo Seixs Prdo 1,2,3,4, Crlos Dniel Llos 1, Anil Mglhães-Neto 1,2,5 nd Luiz-Cludio Cmeron 1,2,6 * 1 Lortory of Protein Biohemistry, Federl University of Stte of Rio de Jneiro, Av. Psteur, 296, Ur, Rio de Jneiro, Brzil 2 Institute of Genetis nd Biohemistry, Federl University of Uerlândi, Av. Prá, 172, Umurm, Uerlândi, Brzil 3 Biologil nd Helth Sienes Center, Tirdentes University, Av. Murilo Dnts, S/N, Frolândi, Arju, Brzil 4 Poet João Freire Rieiro Shool, Sergipe Seretry of Edution, Av. Nção, S/N, Jrdins, Arju, Brzil 5 Biologil nd Helth Sienes Institute Federl University of Mto Grosso, Rodovi MT-1, Km 3,5, Pontl do Argui, Mto Grosso, Brzil 6 Grdute Progrm in Humn Movement Siene University Cstelo Brno RJ Brzil Av. Slvdor Allende, 6.7, Rereio Rio de Jneiro, Brzil (Reeived 17 Novemer 29 Revised 11 My 21 Aepted 12 My 21) During exerise, mmoni levels re relted to the pperne of oth entrl nd peripherl ftigue. Therefore, ontrolling the inrese in mmoni levels is n importnt strtegy in meliorting the metoli response to exerise nd in improving thleti performne. Free mino ids n e used s sustrtes for ATP synthesis tht produes mmoni s side produt. Keto nlogues t in n opposite wy, eing used to synthesise mino ids whilst deresing free mmoni in the lood. Adult mle rts were divided into four groups sed on reeiving either keto nlogues ssoited with mino ids (KAAA) or pleo nd resistne exerise or no exerise. There ws n pproximtely 4 % inrese in mmonemi due to KAAA supplementtion in resting nimls. Exerise inresed mmoni levels twofold with respet to the ontrol, with smller inrese (out 2 %) in mmoni levels due to exerise. Exerise itself uses signifint inrese in lood ure levels (17 %). However, KAAA redued lood ure levels to 75 % of the pre-exerise vlues. Blood urte levels inresed 28 % in the KAAA group, independent of exerise. Supplementtion inresed gluose levels y 1 % ompred with ontrol nimls. Exerise did not hnge gluose levels in either the ontrol or supplemented groups. Exerise promoted 57 % inrese in ltte levels in the ontrol group. Supplementtion promoted twofold exerise-indued inrese in lood ltte levels. The present results suggest tht n ute supplementtion of KAAA n derese hypermmonemi indued y exerise. Ammoni: Urte: Ure: Resistne exerise Ammoni (NH 3 þ NH 4 þ ) is toxi metolite with deleterious effets on the entrl nervous system (1). Exerise n e used s model to study mmoni metolism in n intensitydependent wy (2 5). During prolonged exerise, mmoni is minly produed y the tolism of mino ids (6).Onthe other hnd, during high-intensity exerise, the lrgest soure of mmoni prodution is from AMP demintion (7). Ammoni levels re relted to the pperne of oth entrl nd peripherl ftigue (8). Therefore, ontrolling inreses in mmoni is n importnt strtegy in meliorting the metoli response to exerise nd in improving thleti performne (9,1). The omintion of keto nlogues with mino ids hs een used to tret ptients with hroni kidney disese (CKD), portl systemi enephlopthy nd hypermmonemi (11,12). Free mino ids n e used s sustrtes for ATP synthesis, whih produes mmoni s side produt (13). In n opposite mnner, the use of keto nlogues ssoited with mino ids (KAAA) hs een proposed s wy to synthesise mino ids whilst deresing free mmonemi (14,15). During metolism, mino ids re deminted or trnsminted to form keto ids vi relese of the mino group (16). These retions re reversile, nd the use of keto nlogues ould redue the lood mmoni onentrtion, resulting in the prodution of mino ids (17). Thus, keto nlogues my serve s nutritionl supplements to synthesise mino ids of high iologil vlue, espeilly in CKD ptients. Furthermore, it is knowledged tht resistne nd eroi exerise progrmmes my serve importnt roles in the pproh to the tretment, prevention nd slowed progression of CKD (18). Although KAAA supplementtion is effetive in the tretment of CKD, prtiulrly for postponing the neessity for dilysis, the use of KAAA is not populr due to its ost nd Arevitions: CKD, hroni kidney disese; Ctl group, ontrol group (neither keto nlogues ssoited with mino ids nor exerise); Ex group, exerise-only group; KA group, keto nlogues ssoited with mino ids-only group; KAAA, keto nlogues ssoited with mino ids; KAEx group, keto nlogues ssoited with mino ids nd exerise group. * Corresponding uthor: Professor L.-C. Cmeron, emil meron@unirio.r

2 2 R. D. de Almeid et l. the requirement for low protein intke (19). Thus, there hs een lk of interest in studies involving KAAA due to the lk of ost effetiveness in its use s therpeuti gent. This lk of interest hs limited the numer of new ppers pulished on the mehnism of tion of KAAA. In the present study, we evluted the effet of KAAA supplementtion on mmoni prodution nd lood ure levels during resistne exerise, showing metoli effets tht n enhne performne nd post-exerise reovery. Mterils nd methods Mle Wistr rts (12 weeks of ge nd ody mss rnging from 28 to 35 g) were divided into four groups of twelve nimls eh. The group tht reeived only KAAA (KA group) nd the group tht reeived KAAA nd exerise (KAEx group) reeived 1 g Ketosteril w (Fresenius Ki, Bd Homurg, Germny) in 5 ml wter ( 3 g/kg). The omposition of the KAAA mixture per tlet ws s follows: -keto nlogues of isoleuine, 335 mg; leuine, 55 mg; phenyllnine, 43 mg; vline, 34 mg; -hydroxy nlogue of methionine, 295 mg; L-lysine ette, 75 mg; L-threonine, 265 mg; L-tryptophn, 115 mg; L-histidine, 19 mg; L-tyrosine, 15 mg. The group tht reeived neither KAAA nor exerise (ontrol (Ctl) group) nd the group tht reeived only exerise (Ex group) reeived 5 ml of 9 % NCl, 1 h efore exerise y orl dministrtion. The nimls were mintined in olletive ges (four per ge) t 22 ^ 28C with photoperiod of 12 h nd fed d liitum (diet nd wter). The study ws pproved y the Ethis Committee in Reserh of the University of Tirdentes, nd followed the Guiding Priniples for Reserh Involving Animls nd Humn Beings. Resistne exerise ws performed ording to previous study (2) fter fmiliristion nd determintion of the lod tht ws to e pplied ording to the one repetition mximum (1RM) test. Fmiliristion onsisted of tthing the niml to the exerise devie dily without stimulting the nimls to exerise, strting 6 d efore the experiment. The 1RM test ws performed 1 d efore resistne exerise nd determined the heviest weight tht ould e lifted. On the dy of the experiment, fifty repetitions were performed with lod equl to 75 % of 1RM. The nimls were stimulted to perform the repetitions through stiker eletrodes (Axelgrd VluTrode CF32; Axelgrd Mnufturing Co. Ltd, Fllrook, CA, USA) pled in the til nd onneted to n eletrostimultor (4 ma to 15 ma t 1 Hz for 1 s; Qurk Dulpex 961; Qurk Medil Produts, São Pulo, Brzil). Blood ws olleted through rdi punture efore exerise (Ctl nd KA groups) or immeditely fter exerise (Ex nd KAEx groups). The lood smples were immeditely entrifuged to otin ser, whih ws susequently frozen nd stored t 278C for future iohemil nlysis. Biohemil nlyses of gluose, ure, urte nd retinine onentrtions were performed using ommerilly ville spetrophotometri ssys (Ltest, Mins Geris, Brzil). Ltte nd mmoni were mesured using n enzymi UV method (Rndox, Crumlin, Co. Antrim, UK) on Dde Model Dimension RXL Automted Chemistry Anlyzer (Dde Behring, Eshorn, Germny), nd hemtologil prmeters were nlysed using Sysmex SE-95 Automted Hemtology Anlyzer (TOA Medil Eletronis, Koe, Jpn). Stndrd urves were tken t minimum r vlue of 98 nd the experimentl points were lwys within the lirtion urve nd t lest 2 % ove the lower limit of detetion. Sttistil signifine ws evluted y one-wy ANOVA. Signifines (P, 5) were onfirmed using the Tukey test s post ho nlysis. Dt re reported s men vlues with their stndrd errors. Results We used weight-lifting exerise to evlute the effet of KAAA on lood mmoni onentrtion fter resistne (A) 2 Ammoni (µmol/l) (B) Ure (mmol/l) (C) Urte (µmol/l) Inrese in mmonemi (%) Ctl Ex/Ctl KAEx/KA d KA Ex KAEx Ctl KA Ex KAEx Ctl KA Ex KAEx Fig. 1. Aute supplementtion of keto nlogues ssoited with mino ids (KAAA) ffets mmoni (A), ure (B) nd urte (C) metolism. Ctl, ontrol group (neither KAAA nor exerise); KA, KAAA-only group; Ex, exerise-only group; KAEx, KAAA nd exerise group. Vlues re mens, with stndrd errors represented y vertil rs.,,,d Men vlues with unlike letters were signifintly different (P, 5). Inset of Fig. 1(A) shows the inrese in mmoni levels in experimentl nd ontrol groups when normlised ginst rest vlues.

3 Keto nlogues redue mmonemi in exerise 3 exerise. There ws n inrese of out 4 % in mmoni in resting nimls due to KAAA supplementtion. Compred with unsupplemented ontrols, exerise resulted in twofold inrese in mmoni levels in the nimls. However, the supplemented group hd muh smller inrese (out 2 %) in mmoni levels fter exerise (Fig. 1(A)). Sine KAAA is proposed to derese lood ure levels, we evluted the response of lood ure to ute KAAA supplementtion during exerise. We did not mesure the effet of supplementtion on lood ure t rest. With no supplementtion, exerise signifintly inresed lood ure levels y 17 % ompred with the levels in the Ctl group. However, with KAAA supplementtion, lood ure ws redued to 75 % of the pre-exerise vlues (Fig. 1(B)). To differentite the mmoni prodution derived from AMP demintion from tht derived from mino id demintion, we mesured urte, the end metolite of inosine monophosphte. Blood urte levels inresed 28 % in the group supplemented with KAAA, independent of exerise. This effet ws enhned in the supplemented group fter exerise. There ws no hnge in lood urte levels in the ontrol group in response to exerise (Fig. 1(C)). KAAA supplementtion hs een shown to inrese retinine lerne. Our study model did not detet hnge in lood retinine in response to exerise. However, lood retinine deresed y 4 % in the groups supplemented with KAAA, independent of exerise (Tle 1). To understnd the effet of KAAA on gluose mintenne, we mesured gluose levels fter exerise. Supplementtion inresed gluose levels in resting nimls y 1 %. Exerise did not hnge gluose levels in either the Ctl or KAEx groups (Tle 1). Blood ltte is n inditor of gluose utilistion during exerise. Exerise promoted 57 % inrese in lood ltte in the Ctl group. The supplementtion promoted twofold exerise-indued inrese in lood ltte (Tle 1). Disussion It is widely reported tht mmoni prodution inreses during exerise nd tht mmoni ould e deleterious metolite tht promotes ftigue (8,2,21). The prodution of mmoni n led to signifintly elevted systemi mmoni levels to levels etween 9 nd. 2 mmol/l. Ptients with either liver or kidney disese lso show shrp inreses in mmoni levels tht my rnge from 7 to 3 mmol/l in liver disorder ptients (1,22). Ptients with CKD hve lower pek levels of mmoni during exerise, ut experiene mmoni inreses of out 3 6 % ompred with resting vlues (1,23,24). KAAA hs een widely used s supplement to tret ptients with kidney filure nd s therpeuti gent for liver filure nd enephlopthy (12). Additionlly, regulr physil tivity nd lose linil nd dietry monitoring, inluding the use of keto nlogues, should e reommended in ptients with CKD (25). One of the prolems ssoited with humn studies hs een ensuring tht sujets hve dhered to the reommended diet nd hve properly tken the supplements. Here, we used previously desried resistne exerise niml method (19) to investigte possile mmoni-helting effet of KAAA during exerise in rts. The prodution of mmoni during exerise ours vi oth AMP demintion nd rnhedhin mino id metolism (1). The use of KAAA inresed mmonemi during the resting stte, demonstrting tht mino id metolism during exerise is ssoited with nplerosis of Kres yle intermedites (26,27). Inreses in mmoni levels in response to exerise n e mnged through the use of mino ids or rohydrtes tht interfere with mmoni metolism (28).It is possile to propose tht the mino ids in the supplement re eing using either s ron skeleton donors to otin energy or s gluoneogeni preursors. Even with n inrese in mmoni levels t rest, KAAA supplementtion ws le to redue the exerise-indued inrese in lood mmoni y 8 %. When ompred with the non-supplemented exerise group, the solute derese ws 2 %. Previous dt in our lortory showed tht there is hitution of sl mmoni levels in response to mino id supplementtion, sine the resting mmoni level dereses with n inrese in sl lood ure levels orrelted to supplementtion time (3).On the sis of these dt, we postulte tht the effet of KAAA supplementtion on sl mmoni levels n e diminished y hroni KAAA use. Our exerise model inresed mmoni nd ure levels in nimls without ny hnges in urte levels. It hs een pointed out tht exess mmoni is metolised to ure y the liver for exretion to minimise toxiity (2). During exerise, KAAA ws le to derese the lood ure onentrtion to 75 % of the resting ure level. This finding is relted to the widely desried therpeuti effet of KAAA (for review, see Svi et l. (13) ). Urte ppers more quikly in lood in response to exerise ompred with ure (29). KAAA supplementtion inresed resting urte levels. However, we deteted hnges in lood urte in the supplemented exerise group when ompred with the non-supplemented exerise group fter resistne exerise. It is known tht during Tle 1. Cretinine, gluose nd ltte s otined from the four supplementtion protools (Men vlues with their stndrd errors) Group... Ctl KA Ex KAEx Prmeter Men SE Men SE Men SE Men SE Cretinine (mmol/l) Gluose (mmol/l) Ltte (mmol/l) Ctl, ontrol (no keto nlogues ssoited with mino ids or exerise); KA, keto nlogues ssoited with mino ids only; Ex, exerise only; KAEx, keto nlogues ssoited with mino ids nd exerise.,, Men vlues within row with unlike supersript letters were signifintly different (P, 5; ANOVA).

4 4 R. D. de Almeid et l. high-intensity exerise suh s resistne exerise, the lrgest quntity of urte is produed when the ATP:ADP rtio dereses whih leds to inreses in oth AMP demintion nd urte synthesis (8). It is possile tht our resistne exerise model with fifty repetitions tivtes pthwys ssoited with resistne nd prolonged exerise. KAAA supplementtion promoted n inrese in retinine lerne. This is welldesried effet of this supplement in hronilly ill ptients (12). Tking these results together, we postulte tht the mjority of mmoni prodution results from the demintion of mino ids insted of AMP (7,8,3). Some studies hve shown tht mino id supplementtion inresed the pool of Kres yle intermedites during exerise (5,31). KAAA supplementtion produed 1 % inrese in resting gluose levels tht were mintined even fter exerise. Sine KAAA is mixture of ketogeni nd gluogeni keto nlogues nd mino ids, we postulte tht KAAA provide gluose for exerise. It is importnt to stte tht the use of the mino ids from KAAA s ron skeleton donors ugments the net mmoni relese. On the other hnd, the nplerosis using the keto nlogues does not inrese mmoni relese. However, oth situtions inrese ATP synthesis, leding to deelerting mmoni prodution due to AMP demintion. The results of the present study showed tht KAAA supplementtion exerted lood ltte levels fter exerise. It is known tht ltte is formed during glyolysis in tive skeletl musles nd mny onditions n ttenute ltte levels during exerise, suh s musle glyogen depletion (32). Thus, suh ltertions in the present study my e explined y KAAA providing gluose for exerise through gluoneogenesis. Sine the entrl nervous system hs no effetive ure yle nd depends on the synthesis of glutmine for removl of the exess mmoni (9,33), high levels of lood mmoni hve een proposed to e relted to the development of oth lol nd entrl ftigue (9,21,34). 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