A concept of dietary dipeptides: a step to resolve the problem of amino acid availability in the early life of vertebrates

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1 The Journl of Experimentl Biology 8, Pulished y The Compny of Biologists 5 doi:./je A onept of dietry dipeptides: step to resolve the prolem of mino id vilility in the erly life of vertertes Konrd Drowski, *, Bendik F. Terjesen, Yongfng Zhng,, Jmes M. Phng 5 nd Kyeong-Jun Lee,6 Shool of Nturl Resoures, The Ohio Stte University, Columus, OH, USA, Institute of Aquulture Reserh nd Aquulture Protein Centre CoE, Protein nd Amino Aid Setion, Sunndlsør, N-66, Norwy, The Ohio Stte University Interdisiplinry Progrm in Nutrition, Columus, OH, USA, 5 Metolism nd Cner Suseptiility Setion, Lortory of Comprtive Crinogenesis, Ntionl Cner Institute, Frederik, MD 7, USA nd 6 Fulty of Applied Mrine Siene, Cheju Ntionl University, Jeju , South Kore *Author for orrespondene (e-mil: drowski.@osu.edu) Aepted 7 My 5 The premise tht dietry dipeptide pproh will improve the understnding of mino id utiliztion in the fstest-growing verterte, the teleost fish, ws tested y exmining the musle free mino id (FAA) pool nd enzyme tivities, in onert with growth response, when dietry mino ids were provided in free, dipeptide or protein moleulr forms. We present the first evidene in fish tht, in response to syntheti dipeptide diet, musle FAA vries s result of oth growth rte nd mino id vilility of speifi peptides. We demonstrte signifintly diminished musle indispensle FAA ( - fold) in rinow trout levins fed dipeptide-sed diet ompred with protein-sed diet. The dipeptide-sed diet did not ontin proline, resulting in 7-fold less musle free proline nd hydroxyproline in levins. The response of levins fed FAA-sed or peptide-sed diets n e inditive of ollgen turnover (Hyp/Pro rtio) nd Summry showed signifint differenes etween dietry tretments. Pyrroline-5-roxylte (P5C) redutse tivity ws deteted, suggesting tht P5C my meliorte proline defiieny, ut synthesis from glutmte ould not mintin free proline levels in musle. This finding will provide n impetus to test whether proline is onditionlly indispensle in young fish, s in mmmls nd irds. This study shows tht mino ids given entirely s dipeptides n sustin fish growth, result in musle FAA nd enzyme responses in line with dietry levels nd identify growth-limiting mino ids. The understnding of these ftors neessittes diet formultion tht will improve the ury of determining mino id requirements in the erly life stges of vertertes. Key words: dipeptide, protein, mino id, proline, teleost, rinow trout, Onorhynhus mykiss. Introdution One of the most disputed onepts of digestive physiology t present is the role of dipeptides in retion of indispensle mino ids (IDAA) in niml nd humn nutrition in sttes of helth nd disese (Fürst nd Kuhn, ). As mentioned in the ove review, the dipeptides re innovtive, true, new sustrtes destined to prepre omplete, well lned diets tht will eome solution to prolems in the field of linil nutrition. We propose to use teleost lrvl fish s miniture verterte model in vivo euse of their unique fetures demonstrted in vitro (see Verri et l., ) in order to understnd peptide mino id trnsport nd regultions of sorption mehnisms. Requirements of protein for mximum growth in teleost lrvl nd juvenile stges re nerly twie s high s in older fish (Drowski, 986). The reson for this my e due to underestimted IDAA requirements euse of suoptiml growth rtes in juvenile fish when purified diets were used (Drowski, 986; NRC, 99). There is essentilly no dt for lrvl, pre-metmorphosed fish mino id requirements euse of limittions in formulting eptle diets. Determintion of tissue free mino id (FAA) levels during requirement studies or omprison with dietry mino id omposition hs met with vrile suess in fish (Shumher et l., 997; Ymmoto et l., ). However, most of these mesurements hve een mde for plsm, while in fish the white musulture is quntittively the most importnt site for protein retion (Crter nd Houlihn, ). In humns, musle FAAs hve een found to vry ording to dietry levels nd pthologil nd toli onditions (Fürst nd Stehle, ). Ontogeneti, dietry nd post-prndil effets on

2 886 K. Drowski nd others musle FAAs re lso importnt in rinow trout (Crter et l., 995). We doumented previously tht syntheti dipeptide-sed diet supported growth of rinow trout levins (first-feeding) wheres free mino id mixture diet did not (Drowski et l., ). Hydrolystes s soures of peptides re insuffiient, sine suh peptides re impired y their hrteristis, i.e. the rnge of moleulr sizes nd diffiulties in ontrolling their mino id omposition in requirement studies. There is evidene tht tetr- nd lrger peptides, in the sene of pnreti enzymes nd defiienies of rush order peptidse tivity, eome inple of overing nitrogen requirements (Grimle, 99; Dniel, ). Losses of IDAA during hydrolyste preprtion re frequently responsile for nutritionl indequies resulting in growth depression (Lngr et l., 99). This my hve een the se when the proportion of hydrolyste ws higher thn % protein replement in lrvl fish diets (Chu et l., 999). Advntges of using speifi di- or tripeptides s sustrtes for prtiulr peptide trnsporters hve een determined sed on the ffinity of PEPT trnsporters (Doring et l., 998). These trnsporters were found to e expressed in lrvl teleosts prior to the first exogenous feeding (Verri et l., ). We hypothesize tht syntheti dipeptide diets n e instrumentl in defining mino id requirements for pre-metmorphosed fish, whih is lrgely unknown. As first step, we set out to determine whether physiologil indies suh s musle FAA nd enzyme tivities would respond to suh diets, in omprison with dietry mixture of syntheti mino ids or other moleulr forms. The speifi mino id omposition of the dipeptide diets ws sed on the pity of ytosoli peptidses in fish intestinl epithelil ells (Arnishi et l., 998) nd on known IDAA requirements for rinow trout juveniles (NRC, 99). A series of feeding trils ws rried out with first-feeding levins nd lrger, juvenile rinow trout (Drowski et l., ). We hypothesized tht the onentrtions of FAA in fish musle (onstituting 65 8% of ody mss; Wetherley nd Gill, 987) would e n integrted mesure of vilility of dietry mino ids for protein synthesis nd, t the sme time, n indition of protein synthesis nd degrdtion rtes. Although not ntiipted, we disovered tht free proline onentrtion in musle showed strong dietry dependeny nd ws possily onditionl indispensle mino id, similr to in young mmmls (Kirhgessner et l., 995). We followed this line of inquiry nd, for the first time, nlyzed pyrroline-5-roxylte redutse (P5CR) tivity in fish, the finl step in proline iosynthesis. Mterils nd methods Feeding trils The design of the experiments nd diet formultions where tissue smples for FAA nd enzyme nlysis were olleted were desried erlier (Drowski et l., ). Briefly, dietry mino ids were provided in free, peptide or protein moleulr forms (Tle ). Diets were isonitrogenous nd isolipidi nd ontined either () sein-geltin s the protein soure [ sein diets, with (+) or without ( ) inlusion of m mel; Lee et l., ], () mixture of syntheti dipeptides ( dipeptide diet) or () mixture of syntheti mino ids ( diet). The levels of mino ids in the experimentl diets (g g ) were lulted from dded mounts (Drowski et l., ), Sigm (St Louis, MO, USA) nd ICN Tle. Compositions (%) of the four experimentl diets (modified fter Drowski et l., ) Ingredients/diets Csein( ) Csein(+) Csein.. Geltin mix.6 mix.6 Whet mel 5. M mel Dextrin CPSP Cod liver oil.... Vitmin mix.... Minerl mix Vitmin C CMC L-Arg L-Met.... L-Lys Choline hloride.... Cellulose.. Csein( ): sein-geltin s the protein soure. Csein(+) is the sme diet s sein( ) exept for the inlusion of m mel. : mixture of syntheti dipeptides. : mixture of syntheti mino ids. mix omposition (g.6 g ; ll L-form AA unless otherwise indited): Arg,.5; His,.7; Ile,.9; Leu,.9; Lys,.8; Met,.; Phe,.8; Thr,.8; Trp,.; Vl,.; Pro,.; Ser,.; DL-Al,.. mix omposition (g.6 g ): Arg Vl,.87; His Leu,.; Gly Ile,.; Lys Gly,.96; Gly Met,.8; Phe Leu,.; Thr Leu,.56; Gly Trp,.6; Vl Leu,.5; Al Gly, ; Al Gln, 5; Gly Tyr,.95. Solule fish protein hydrolyste; Sopropehe S. A., Boulogne Sur Mer, Frne. Rohe Performne Premix omposition (g kg of vitmin mixture): vitmin A ette, 7.56; holeliferol,.55; α- toopheryl ette, 66.; vitmin B,.; rioflvin,.; niin, 6.7; d-pntotheni id,.; mendione,.; foli id,.76; pyridoxine,.; thimin, 7.95; d-iotin,. (Hoffmn-L Rohe, In., Nutley, NJ). 5 Five mg Se in the form of sodium selenite per kg Bernhrt Tomrelli slt mixture (ICN Phrmeutils In., Cost Mes, CA, USA). 6 Mg-L-soryl--phosphte; Show Denko K. K., Tokyo, Jpn. 7 Croxymethylellulose; ICN Biomedils, In., Cost Mes, CA, USA.

3 Amino id vilility in juvenile fish 887 Biomedils, In. (Cost Mes, CA, USA) produt sheets of dietry ingredients, nd literture (Kim et l., 99; Berge nd Storekken, 996; Ymmoto et l., ; Hlver, ). All diets overed the requirements for IDAA in rinow trout (NRC, 99). Experiment Rinow trout Onorhynhus mykiss Wlum of lol strin (London, OH, USA) were hthed nd, t the firstfeeding stge (±6 mg wet mss individul ), distriuted rndomly into triplite tnks for eh dietry tretment. After 5 weeks of feeding, fish were olleted from eh tnk nd trnsferred into n ie wter th nd then individully frozen on dry ie nd stored t 8 C for FAA nlyses. Experiment Juvenile rinow trout of.78±.8 g initil mss individul nd pproximtely 5 weeks old were used. Three diets were tested:, dipeptide nd sein( ). At the ompletion of the -week experiment, smpling tissues for FAA nd P5CR tivity nlyses ommened in the morning, h fter the lst feeding, nd were onduted rndomly ross tnks nd tretments. Fish were killed nd smples stored in the sme mnner s in Experiment. Experiment This experiment imed to determine hnges in P5CR tivity with time durtion of the ssy, i.e. to optimize kinetis. Furthermore, it imed to nlyze tissue distriution of P5CR tivity etween week nd 6 fter the first feeding. Thus, it would inlude the time-periods of rinow trout ontogeny studied in Experiments nd. Rinow trout levins were rered s desried erlier nd fed sein-sed diet (% sein, 6% geltin, 6% sein-hydrolyste; K. Drowski nd M. Penn, personl ommunition) until smpling t weeks nd 6. Fish were olleted from eh tnk t smpling nd individully frozen in liquid nitrogen nd stored t 8 C. To estlish the ssy onditions nd presene of P5CR tivity in fish, we used fresh tissues nd individuls of lrger size to seprte orgns of interest: liver nd intestine. Tissue smples of -month-old rinow trout were therefore ssyed. These fish were fed stndrd ommeril diet (Zeigler-Bros., Grdners, PA, USA). After 8 h fsting, four fish (5± g individul, 7.6±.5 m length) were killed y shrp low to the hed, nd the intestine (inluding pylori e) nd the liver disseted out, rinsed in.9% NCl nd ssyed immeditely. FAA nlysis Individul fish from Experiments nd were rpidly disseted while still frozen on ie-ooled ords. The hed nd til (eyond the dominl vity) were removed, nd the dorsl ody region, nterior nd posterior to the dorsl fin, ws disseted out (8±8% of ody mss; N=76). Cre ws tken to void remins of kidney tissue y srping ny lood ontining tissue from the upper prt of the ody vity. The tissue smples were weighed nd re-frozen t 8 C. Thus, these musle tissues lso ontined skin, rtilge nd one, ut were lrgely omposed of white musle, nd re, for simpliity, termed musle. Within two dys, musle smples were extrted in. mol l HCl ontining 6 μmol l norleuine, using tissue:extrtion medium rtio of : (juveniles) or : (levins) (w/v), ording to Cohen et l. (989). The norleuine reovery ws ±8% (N=5). Tissue extrts were spun t g ( C, 5 min), nd superntnts filtered (Millipore, Billeri, MA, USA; kd ut-off t g, C, 9 min). Smples of lnks nd externl stndrds (Sigm id/neutrl nd si mino ids), supplemented with glutmine, were prepred t the sme time s smple extrtion y djusting ppropritely with distilled nd deionized H O, nd. mol l HCl ontining 6 μmol l norleuine. Tissue extrts, stndrds nd lnks were then stored t 8 C, for lter nlysis using the Wters (Milford, MA, USA) PioTg method with pre-olumn derivtiztion nd reverse-phse high-performne liquid hromtogrphy (RP-HPLC; Cohen et l., 989). Enzyme tivity P5CR (EC.5..) tivity ws mesured using modifitions of mmmlin protools (Herzfeld et l., 977; Dekney et l., ). DL-P5C ws produed from its,- dinitrophenylhydrzone (Sigm), purified y tion-exhnge hromtogrphy, nlyzed with o-minoenzldehyde nd stored t C in.5 mol l HCl (Mezl nd Knox, 977). Liver nd intestine from juvenile trout (ssys in fresh tissues), or liver nd intestine from levins or juveniles from Experiment (N= tnks), for P5CR tivity nlysis were otined using dissetion tehniques desried ove. The whole intestine, inluding pylori e nd pnres, ws used. Liver onstituted %, nd intestine 8 9%, of totl ody mss, nd no hnges in hepti or intestinl indies were found when ompring Experiment levins fed for weeks (.7±. g ody mss) with those fed for 6 weeks (.8±.9 g ody mss). To ssess totl P5CR tivity in juvenile trout fed the different experimentl diets (Experiment ), ssys were run on the whole fish ody (Terjesen et l., ). Tissues to e ssyed for P5CR tivity were homogenized on ie (Potter- Elvehjem t rev min, 6 s, two strokes) in 5 mmol l surose, 5 mmol l phosphte uffer (ph 7.), mmol l EDTA,.6 mmol l dithiothreitol, nd.5 μg ml eh of protinin, pepsttin A, hymosttin nd phenyl methyl sulphonyl fluoride. Whole-ody smples were gently disrupted prior to the Potter-Elvehjem y n Omni GLH homogenizer ( rev min, 5 s). All extrts were spun t 6 g ( min, C), the superntnt further entrifuged t 5 g ( min, C), nd the finl superntnt used for mesurements. This pproh (Dekney et l., ) ws followed ssuming tht P5CR hs ytosoli lotion in fishes, s in mmmls. Proline oxidse, whih ould utilize the P5CR retion produt, hs mitohondril lotion in mmmls (Wu nd Morris, 998). Immeditely efore ssy, the P5C sustrte ws neutrlized

4 888 K. Drowski nd others on ie, using mol l Hepes (ph 7.) nd mol l NOH. Duplite retions were run in μl totl volume ( μl extrt), ontining.5 mmol l DL-P5C, mmol l NADH nd 5 mmol l phosphte uffer (ph 6.8). After 5 min t 6 C (Terjesen et l., ), retions were terminted y 5 μl of.5 mol l HClO nd neutrlized with.5 μl of mol l K CO. After entrifugtion (6 g), proline ws determined y PioTg RP-HPLC (Cohen et l., 989), nd protein determined y the Brdford method (Brdford, 976). The mount of proline formed ws liner with time nd protein onentrtions, nd ontrol ssys without P5C or enzyme extrt did not result in detetle tivity (hromtogrms not shown). Dt nlysis Dt re presented s mens ± S.D. Tnk mens were used s the sttistil unit, nd dt were nlyzed in SPSS version... (SPSS In., Chigo, IL, USA) using one-wy nlysis of vrine (ANOVA) for ll tests exept for Experiment regrding P5CR tissue distriution nd ge sine first-feeding (two-wy ANOVA). If signifint (P<.5), Dunn s multiple rnge tests were employed. Results Exept for feed prtile size, the speifi diets given to oth life stges were identil nd were rpidly ingested (Drowski et l., ). Rinow trout showed life-stge-speifi musle FAA responses to dietry mino ids (Figs, ). Rinow trout levins (Experiment ) fed or dipeptide diets hd signifintly lower totl FAA musle onentrtions thn levins fed the sein(+/ ) diets (Tle ). The sum of dispensle mino id (DAA) ws twie s high in juveniles (Experiment ) ompred with levins fed the diet, wheres the IDAA onentrtions were not signifintly different etween the life stges (Tles, ). Conerning speifi FAA nd differenes etween the life stges, the high lysine onentrtions in levins fed the diet (Tle ) my hve refleted diminished protein synthesis sine it orresponded to low growth rte (Fig. ) in omprison with juveniles (Fig. ; Tle ). The high proline level in the Free AA diet did not result in elevted free proline in musle of levins (Fig. C). This ws in ontrst to musle of juvenile trout fed the diet (Fig. C). Similrly, the dipeptide diet, rih in glyine, resulted in only modest level of free glyine in rinow trout levins (Fig. A), wheres the sme diet mounted to mmol kg wet mss glyine in musle of juveniles, the highest level of single FAA (Fig. A). Severl FAAs in musle, suh s histidine, threonine, vline, rginine nd lnine, orresponded to dietry mino id profiles in Experiments nd. The sein(+/ ) diets hd higher IDAA level thn the nd dipeptide diets, nd this ws mirrored in oth levin nd juvenile musle FAA onentrtions (Figs B, B; Tles, ), nd resulted in the highest growth rtes (Figs,, top digrm). However, these differenes in musle free IDAA, e.g. histidine ( 8-fold), were in mny ses severl folds lrger thn the dietry level differenes (Figs B, B; Tles, ). Among free DAA, lnine, whih ws high in the diet, rehed 8 mmol kg wet mss in musle of juveniles (Fig. A), nd lnine onentrtions were signifintly higher in fish of oth life stges fed the diet ompred with trout fed other diets. Free sprti or glutmi ids in musle did not indite ny impt of dietry levels (Figs A, A). The musle onentrtions of severl free IDAA were ffeted y dietry moleulr form. In prtiulr, isoleuine nd methionine were present in musle t higher levels when given diet, in oth levin (Experiment ) nd juvenile trout (Experiment ), ompred with the dipeptide diet fed fish, despite similr dietry levels (Figs B, B; Tles, ). This oservtion my imply tht n exess of mino ids ws not utilized for protein synthesis in the Free AA diet fed levins (Experiment ), sine negligile weight gins (Fig. ) nd elevted mmoni exretion were oserved (Drowski et l., ). To the ontrry, there ws no signifint differene in mmoni exretion rte in juveniles fed nd sein( ) diets, nd improved, lthough low, weight gin ws oserved in juveniles fed diet (Fig. ; Drowski et l., ). The ssys of fresh rinow trout juvenile tissues showed tht P5CR tivity ws present in liver nd intestine (dt not shown). P5CR tivity ws present in the intestine nd liver of rinow trout levins fed for weeks (Fig. A). Liver hd signifintly higher P5CR tivity thn intestine (P<.), ut no signifint effets of ge, or ge tissue, were noted (twowy ANOVA). The effets of different dietry soures of mino ids on P5CR tivity were investigted in Experiment (Fig. B). The juvenile trout fed the dipeptide diet showed numerilly higher P5CR tivity thn juveniles fed nd sein-sed diets, lthough differenes were not signifint (P=.5; one-wy ANOVA). When the totl P5CR tivity in the whole ody ws estimted in the sein-diet-fed trout (.7 U g wet mss) nd ompred with the tivity of intestine (. U g wet mss) nd liver (. U g wet mss), it ws lulted tht these two tissues (% of ody mss) ounted for ~% of whole-ody P5CR tivity. In onlusion, rinow trout levins nd juveniles expressed Fig.. Musle free mino id (FAA) onentrtions in rinow trout levins sujeted to Experiment tretments. (A) Exmples of dispensle mino ids (DAA) in reltion to dietry levels. (B) Exmples of indispensle mino ids (IDAA) suggesting limittions in dietry peptide vilility. (C) Amino ids involved in the proline ornithine rginine pthwy. Dt given s mens ± S.D., two smples of fish eh were ssyed per tnk, three tnks per tretment (N= per group), exept (N=). The top grph (speifi growth rte) is modified from Drowski et l. (). The lower prts of eh figure (rs) show musle onentrtions of individul FAA (y xis title to the left). The upper prts of eh figure (roken lines) indite the level of eh mino id in the diets (g g ; y xis title to the right). Signifint differenes etween tretments (P<.5) re indited y differing letters. Figures with no letters next to the rs indite non-signifint one-wy ANOVAs.

5 Amino id vilility in juvenile fish 889 SGR (% dy ) 6 Growth d Csein ( ) Csein (+) Musle FAA (µmol kg ) Asp Free DAA reltions with dietry levels A B C Csein ( ) Gly Al Tu Csein (+) Thr 6 IDAA suggesting limittions in peptide vilility 5 Csein ( ) Ile Met His Csein (+) Arg Pro Orn Arg pthwy Csein ( ) Hyp.5 Pro, Orn Csein (+).5. 8 Diet AA (g g ) Dietry tretment Fig.. See previous pge for legend.

6 89 K. Drowski nd others SGR (% dy ) Growth Musle FAA (µmol kg ) Asp DAA reltions with dietry levels A B C Gly Al Tu Csein ( ) IDAA suggesting limittions in peptide vilility 5 His Thr 8 6 Csein ( ) Ile Met Csein ( ) Dietry tretment Pro Orn Arg pthwy Hyp Pro, Arg Orn Csein ( ) Diet AA (g g ) Fig.. Musle free mino id onentrtions in rinow trout juveniles sujeted to Experiment tretments. Dt given s mens ± S.D., two smples of fish eh were ssyed per tnk, three tnks per tretment, N= for ll groups. Signifint differenes etween tretments (P<.5) re indited y differing letters. See Fig. for further detils.

7 Amino id vilility in juvenile fish 89 Tle. FAA onentrtions nd seleted rtios in musle of rinow trout levins (Experiment ) not shown in Fig. Dietry tretment Amino id Csein( ) Csein(+) Gln* 67± 85± 858±76 9±9 Glu* ± 5±76 865±77 67±7 Ser* 9±5 99±5 865±87 65± Tyr* 98±8 95± ±56, 56±56 Vl* 7±6 ±7 7±9 5±76 Leu* 6±78 6±8 55±5 99±8 Phe* 6±6 78±8 8± ± Trp* 5± 6±9 67± 7± Lys* 6±7 9± 79±6 88±5 Sum DAA 7.±..±..±..9±.6 Sum IDAA.±.8.8±..±. 8.6±.8 Sum FAA.5±. 7.±..7±.9.±. Orn/Pro.58±..9±.,.±..±. Hyp/Pro.±.7.±.55.7±.77.6±.9 *Dt re given s tnk mens ± S.D. in μmol kg wet musle mss (N=), exept for the diet (N=). Sums of DAA (dispensle mino ids), IDAA (indispensle mino ids) nd totl FAA (free mino ids) (mmol kg wet mss) represent the sum of FAA given here nd in Fig.. Orn/Pro nd Hyp/Pro dt were lulted from vlues given in Fig.. Supersript letters represent signifint effets of dietry tretment; dt within n experiment not shring supersript letter re signifintly different t P<.5. P5CR tivity in liver nd intestine, ut fish ould not mintin musle free proline when fed proline-defiient dipeptide diets (free proline musle levels were -fold lower) in omprison with fish fed proline-ontining sein( ) (juveniles) or oth sein(+) nd ( ) diets (levins) (Figs C, C). Rinow trout levins fed - or dipeptide-sed diets (Experiment ) showed different responses in musle levels of ornithine nd proline nd intertions with growth. Ornithine, whih my e synthesized from P5C, ws higher in levins fed diet thn in levins fed other diets (Fig. C; Tle ), onurrent with negligile growth rte (Fig. ) nd very low proline musle onentrtions (Fig. C), despite eing given diet high in proline. By ontrst, dipeptide-fed levins showed signifintly higher growth rtes thn the FAA-fed groups (Fig. ; Drowski et l., ), nd the dipeptide-fed fish hd omprle musle ornithine levels to tht of the sein(+/ )- fed fish (Fig. C). Despite eing given dipeptide diet, devoid of peptide proline, these fish produed signifintly higher [Orn]/[Pro] rtios (Tle ). In regrd to hydroxyproline, in juveniles (Experiment ) the free musle levels were omprle in the - nd dipeptide-fed groups, ut 7- fold lower thn in fish fed the sein( )-sed diet (Fig. C). In levins (Experiment ), the dipeptide-fed fish (6± μmol kg wet mss) hd numerilly higher free hydroxyproline onentrtion in musle thn did the fed fish (7± μmol kg wet mss); however, this ws 7-fold lower (9± μmol kg wet mss) thn in the fst-growing (.5% dy ) rinow trout levins fed the sein( ) diet (Fig. C). Disussion The onept tht smll peptides re sored in the verterte intestine hs reently een the sujet of mny indepth reviews (Grimle, 99; Dniel, ). However, in vivo evidene of the nutritionl nd metoli signifine of omplete (ll IDAA) peptide diets remins elusive. The finding tht verterte n grow on diet exlusively omposed of syntheti dipeptides of known omposition ws reently reported for the first time in teleost fish y our lortory (Drowski et l., ). There is evidene tht single peptide n e more effiiently sored thn the mixture of identil mino ids oth in fish (Reshkin nd Ahern, 99; Boge et l., ) nd mmmls (Mtthews, 99). The. A.5 B P5CR tivity (µmol proline min mg protein) week week Intestine week week 6 Liver.... Csein ( ) Dietry tretment Fig.. Pyrroline-5-roxylte redutse (P5CR) tivity in rinow trout levins nd juveniles. (A) Liver nd intestinl P5CR tivity in rinow trout levins t weeks nd 6 weeks post first-feeding (Experiment ). (B) P5CR tivity in whole ody of juvenile rinow trout fed mino ids in different moleulr forms (Experiment ). Dt given s mens ± S.D., N= per group. Dt with supersripts indite signifint ANOVA; mens not shring similr letter re signifintly different (P<.5).

8 89 K. Drowski nd others Tle. FAA onentrtions nd seleted rtios in musle of rinow trout juveniles (Experiment ) not shown in Fig. Dietry tretment Amino id Csein( ) Gln* 99± ±5 6±86 Glu* 6± 76±9 95± Ser* 875±6 5±59 ± Tyr* 96± 5±5, 5±6 Vl* ± 7±9 5±9 Leu* 56± ±9 ±96 Phe* ± 69±8 57±8 Trp* ±6 8±, 56± Lys* 6±5 8±579 95±8 Sum DAA 5.6±. 6.±. 7.±.8 Sum IDAA.±..7±.7.±. Sum FAA 9.7±. 9.7±. 8.±.6 Orn/Pro.±..±..±. Hyp/Pro.9±.9.±.5.86±. *Dt re given s tnk mens ± S.D. in μmol kg wet musle mss (N=). Sums of DAA (dispensle mino ids), IDAA (indispensle mino ids) nd totl FAA (free mino ids) (mmol kg wet mss) represent the sum of FAA given here nd in Fig.. Orn/Pro nd Hyp/Pro dt were lulted from vlues given in Fig.. Supersript letters represent signifint effets of dietry tretment; dt within n experiment not shring supersript letter re signifintly different t P<.5. present report provides the first evidene using syntheti dipeptide diets on the regultory mehnisms ffeting mino id sorption nd utiliztion resulting in distint ptterns of musle FAA. In fed nimls, weight gins usully rise s result of inresed protein synthesis nd deresed protein degrdtion in omprison with fsted nimls (Wterlow, 999). Severl ftors re pivotl in determining the effetiveness of these proesses, suh s onentrtions nd orret proportions, i.e. lned mino id omposition to hieve protein retion, i.e. growth. We sumit tht the differenes in musle FAA shown here () re due to differenes in mino id sorption rtes from free, dipeptide or protein dietry soures, () result in uneven umultion rtes nd post-prndil pek times for musulr FAA nd () onsequently result in different metoli hndling of the mino ids nd vilility for protein synthesis. Superimposed on these re differenes in growth rte, protein synthesis rte nd developmentl stges, whih demnd vrying mounts of mino ids t the quntittively most importnt site for protein retion, white musle. This set of ftors, together with enzyme expression dt, provides more insight into mino id nutrition thn nitrogen lne or growth rte lone. As n exmple, methionine ws provided in omprle mounts in ll three diets, ut methionine given in dipeptide form ws present in musle of levins in signifintly lower mounts thn in fish with essentilly no growth ( group) nd higher growth rtes [sein(+/ ) groups], suggesting limittions of the Gly Met dipeptide vilility (Fig. B). A mjor finding in the present study is the strong dependene of free proline in musle tissue on dietry level of proline in dipeptide- nd sein-sed diets (Figs C, C), sine it n e ssumed tht ny exess of proline would e tolized for energy vi P5C nd ornithine or glutmte. In rinow trout juveniles fed dipeptide-sed diet devoid of proline, endogenous synthesis did not mintin proline levels omprle with fish fed diets with proline [sein(+/ )]. The free proline levels in musle of the dipeptide-fed group re t the lower end of the rnge reported in fish musle (Torrissen et l., 99; Ymmoto et l., ; Ogt, ). In lrge rinow trout fed low-protein, 5% sein-ontining diet, only tre mounts of free proline were found in musle, wheres in fish on 5% sein diet, the proline onentrtion ws μmol kg (Yokoym nd Nkzoe, 99). In mmmls, lthough proline n e synthesized from ornithine or glutmte vi P5C, young mmmls, rts nd pigs still require dietry soure of proline for mximum growth nd protein retention (Bll et l., 986). Furthermore, inresed dietry proline levels in young rts nd pigs orrelte with plsm proline onentrtions (Smuels et l., 989; Kirhgessner et l., 995), nd no signifint response ws found in P5CR tivity when ompring pigs fed ontrol nd proline-defiient diets. The findings in young mmmls orrespond to the present study on rinow trout. In fish, - fold drop in the free proline in musle ws ssoited with 8% inrese (ANOVA not signifint) in P5CR tivity in the dipeptide-fed rinow trout, diet devoid of proline (Fig. B). In other fish (Pifi slmon), juveniles fed AA-sed diets were reported for the first time y Hlver et l. (957) to grow t the rte of.5% dy. Tht is similr to rinow trout juveniles in the present study (.8% dy ; Fig. ). The growth dt for fish fed diets with no proline in the Hlver et l. study, however, ended muh erlier thn with other tretments where AA were omitted from the diets. Therefore, results nnot e ompred diretly. Similrly, growth of young tilpi on n AA-sed diet devoid of proline ws shown to e inferior (Aoe et l., 97). However, these uthors disontinued the study. Sine lrger fish were used in most mino id requirement studies, possile proline onditionl indispensility for optiml growth of lrvl fish ws not demonstrted. The lssil study (Hlver nd Shnks, 96) indites tht the finl mss of sokeye slmon fed prolinedefiient diet ws less thn tht of fish fed ontrol diet, nd the experiment ws shortened to only 5 weeks. In ddition, in this previous study (Hlver nd Shnks, 96) nd erlier experiments with Chinook slmon (Hlver et l., 957), proline-defiient diet ws supplemented with more thn generous mount of rginine (.6 5%). Interonversion of rginine to ornithine nd then ornithine to proline in neontl mmmls n reh 5 nd 57%, respetively (Bertolo et l.,

9 Amino id vilility in juvenile fish 89 ). Regrding fish, erly life stges re hrterized y high protein synthesis rtes (7 %), nd protein deposition rtes of 5% ody protein per dy hve een oserved in lrvl tfish (Terjesen et l., 997) nd other speies (Fuonneu et l., 986). Furthermore, during the slttory development of lrvl fish, musle fires nd skeleton undergo onsiderle hnge (Blxter, 988), demnding ollgen synthesis. Sine proline nd hydroxyproline onstitute more thn % of the mino id residues in ollgen in mmmls (Smith nd Phng, 978), nd in fish hydroxyproline lone is estimted t 7% (Sto et l., 989), requirement for dietry proline supplementtion should e investigted in exogenous feeding during the erly life stges. We sumit tht dipeptide-sed diets n e instrumentl in determining dietry requirements for IDAA, inluding onditionl indispensility for proline, in lrvl nd juvenile fish. High onentrtions of hydroxyproline in musle of fstgrowing nimls is n inditor of high rte of ollgen turnover (Adms nd Frnk, 98). Sine hydroxyproline relesed y ollgen rekdown nd susequent hydrolysis of Hyp-ontining peptides is destined for tolism nd exretion, present results in rinow trout re the est indition thus fr of the musle limiting degrdtion of ollgen when there is no dietry supply of proline (Figs C, C). In juvenile slmon, musle hydroxyproline ws the only FAA tht orrelted with growth rtes (Sunde et l., ). Although we indite the presene of hydroxyproline in the sein(+/ )-sed diets, we must ssume tht trnsport of dietry hydroxyproline from intestine to musle is limited (Adms nd Frnk, 98), nd thus high onentrtions in musle of sein/geltin-sed diet-fed fish would reflet high protein turnover rtes. This would suggest tht low proline nd hydroxyproline onentrtions re inditive of either fsting (the diet fed levins) or dietry limittion (the dipeptide diet). We sumit tht this prdox is relted to the only ursory understnding of the proline ornithine rginine pthwy in fish, onditionl indispensility nd interonversion of these mino ids. In onlusion, present oservtions nd dt olleted with the use of dipeptide protein (: rtio)-sed diets (B.F.T. nd K.D., unpulished dt) suggest tht dietry peptide trnsport nd hydrolysis in rinow trout erly life stges n e improved y the inlusion of protein supplement nd more effiiently hydrolysle dipeptides. Consequently, monitoring disproportions of FAA in musle nd expression of glutmte proline ornithine pthwy enzymes, in onert with growth indies, is the right pproh to ddress si mehnisms of mino id utiliztion in lrvl fishes. The modest suess of nutrient dministrtion in the form of dipeptides points to the potentil for evlution of mino id requirements in erly life stges of fishes tht hs not een possile thus fr. We re grteful to G. Wu, Texs A&M University for mmmlin P5CR ssy protools. We knowledge The Ohio Stte University Post-dotorl Fellowship Progrm, nd The Norwegin Reserh Counil (projet no. /599) for funding. Referenes Adms, E. nd Frnk, L. (98). Metolism of proline nd the hydroxyprolines. Annu. Rev. Biohem. 9, 5-6. Aoe, H., Msud, I., Ae, I., Sito, T., Toyod, T. nd Kitmur, S. (97). Nutrition in young rp.. Nutritive vlue of free mino ids. Bull. Jp. So. Si. Fish. 6, 7-. Arnishi, F., Wtne, T., Ostomi, K., Co, M., Hr, K. nd Ishihr, T. (998). Purifition nd hrteriztion of thermostle dipeptidse from rp intestine. J. Mr. Biotehnol. 6, 6-. Bll, R., Atkinson, J. nd Byley, H. (986). Proline s n essentil mino id for the young pig. Br. J. Nutr. 55, Berge, G. M. nd Storekken, T. (996). Fish protein hydrolyzte in strter diets for Atlnti slmon (Slmo slr) fry. Aquulture 5, 5-. Bertolo, R. F. P., Brunton, J. A., Penhrz, P. B. nd Bll, R. O. (). Arginine, ornithine, nd proline interonversion is dependent on smll intestinl metolism in neontl pigs. Am. J. Physiol. Endorinol. 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