Accepted 16 October 2006

Transcription

1 4885 The Journl of Experimentl Biology 29, Published by The Compny of Biologists 26 doi:1.1242/jeb.2597 Postprndil chnges in plsm free mino cid levels obtined simultneously from the heptic portl vein nd the dorsl ort in rinbow trout (Oncorhynchus mykiss) Anders Krlsson 1,2, Erik J. Elison 3, Liv Torunn Mydlnd 1, Anthony P. Frrell 3 nd Anders Kiessling 1,2, * 1 Aquculture Protein Centre (APC), Centre of Excellence nd 2 Deprtment of Animl nd Aquculturl Sciences, Norwegin University of Life Sciences, PO Box 53, N-1432 As, Norwy nd 3 Deprtment of Zoology nd Fculty of Lnd nd Food Systems, University of British Columbi, BC, Vncouver, V6T 1Z4, Cnd *Author for correspondence t ddress 2 (e-mil: nders.kiessling@umb.no) Accepted 16 October 26 For the first time, chnges in plsm concentrtions of free mino cid (AA) nd their metbolites were followed simultneously in pre- nd post-heptic blood following single mel in non-nesthetized nd free-swimming fish. Rinbow trout (Oncorhynchus mykiss), kept in 1 C wter nd fitted with cnnule in the heptic portl vein (HPV) nd the dorsl ort (DA), were force-fed 1% of their body mss nd blood smples were tken from both cnnule t, 3, 6, 12, 24 nd 48 h postprndilly to follow the free AA profile. Almost ll free AAs incresed rpidly within the first 3 h nd only few free AAs did not chnge significntly over time. By 6 h, the totl free AA concentrtion hd peked in blood tken from both the DA (717±369 nmol ml 1 ) nd HPV (9999±572 nmol ml 1 ). However, individul free AAs showed three min profiles beyond this time: for type I, pek concentrtion occurred only t 6 h; for type II, there ws more grdul rise in concentrtion to pek t 24 h; nd for type III there were two peks, t 6 h nd 24 h. All free AAs returned to or were lower thn bseline levels within 48 h, with the Summry exception of threonine nd proline. The totl free AA concentrtions were consistently higher (P<.5) in the HPV thn in the DA t 3 h, 6 h, 12 h nd 24 h. Our dt provide cler evidence tht, during the first pss through the liver, heptic modifiction ltered individul free AA concentrtions s indicted by vrible rtios mong the simultneous blood smples. Furthermore, the elevtion of mmonium nd ure in the HPV indictes intestinl ctbolism of ingested free AA before relese into the HPV. In conclusion, the dul HPV nd DA cnnultion shows promise s useful technique for qulittive nd quntittive investigtions of bsorption nd turnover of nutrients, especilly if the mesurements cn be combined with relible estimtes of blood flow nd lbelled substnces. Key words: Heptic portl vein cnnultion, plsm free mino cids, ure, mmonium, digestibility, protein metbolism, rinbow trout, Oncorhynchus mykiss. Introduction Most reserch into fish nutrition hs involved either growth or digestibility trils (see Hlver nd Hrdy, 22). Although these studies re useful from fish production point of view, they provide little insight into specific rtes of nutrient bsorption vi the intestine nd metbolism by the liver. A few studies hve therefore mde ttempts to follow postprndil nutrient uptke by serilly smpling blood from slughtered fish (reviewed by Crter et l., 21). However, such studies my yield mbiguous results becuse of severl shortcomings. There is the obvious smpling effect of terminl blood smpling techniques which stress fish nd gretly lter gut blood flow (Thorrensen et l., 1993). In ddition, high individul vrition of plsm nutrients my msk nything but mjor chnges. Sunde et l. (Sunde et l., 23) tried to circumvent these obstcles by smpling blood from the dorsl ort (DA) vi permnent cnnul in free-swimming fish. By mesuring postprndil free mino cid (AA) concentrtions in the blood of fish given different diets, they reveled differences in feed qulity not esily discovered with trditionl growth rte-bsed experiments. However, limittion of using DA blood smples is tht the blood metbolites my hve undergone heptic modifiction en route from the intestine to the DA. Blood leving the gut vi the heptic portl vein (HPV) first psses through the liver, nd, given the centrl role of the liver

2 4886 A. Krlsson nd others in the AA metbolism of higher vertebrtes (McDonld et l., 22), differences in the pre- nd post-liver profiles of plsm free AAs re to be expected in fish. In fct, it is estimted tht 2 5% of the free AAs entering the blood never get pst the liver (Hoerr et l., 1991; Hoerr et l.,1993; Biolo et l., 1992; Mtthews et l., 1993). Lyndon et l. tried to estimte the prend post-heptic free AA plsm levels by seril slughter of cod (Gdus morhu) nd smpling of blood from HPV nd crdic puncture immeditely fter deth (Lyndon et l., 1993). Ash et l. (Ash et l., 1998) smpled blood using double DA nd HPV cnnultion technique (McLen nd Ash, 1989) from lightly sedted fish but mde only one mesurement 3 h postprndilly. Thus, to dte relible informtion does not exist on the time course of the postprndil free AA chnges in plsm in fish or on the degree of heptic modifiction of free AA during their first pss through the liver fter uptke by the intestine. By combining the DA nd HPV cnnultion techniques, the present study is first ttempt to follow, over time, the postprndil free AA profiles in plsm simultneously collected before nd fter the liver in nesthetized nd free-swimming fish. Furthermore, by simultneously smpling blood from both the DA nd the HPV, we cn test the hypothesis tht heptic metbolism of free AAs during their first pss through the liver is possible in rinbow trout. Two different dietry tretments were used to ssess the effect of vrying dietry mino cid composition on the free AA plsm profile. In ddition to free AAs, plsm levels of the metbolites ure nd mmonium were nlysed to evlute the potentil for intestinl AA ctbolism. Therefore, the present study provides the first comprehensive study of postprndil AA uptke nd immedite metbolic processing of AAs in fish. Mterils nd methods Rinbow trout Oncorhynchus mykiss Wlbum (verge body mss, 198 g; rnge, g) were plced in six 1 m 3 tnks divided long their digonl xis, with one fish in ech comprtment (N=12). Ech comprtment ws equipped with hide tht consisted of 2 4 mm cover 5 mm bove the wter surfce. Fully erted, well wter (1 C; 2 l min 1 ) ws directed horizontlly to crete stedy current long the outside of the comprtment. Dylight fluorescent tubes provided continuous illumintion. Fish were strved for h prior to surgery. Cnnultion ws performed on nesthetized fish (.1 g l 1 MS-222; tricine methne sulphonte). The DA cnnultion ws performed ccording to the method of Soivio et l. (Soivio et l., 1975) nd included djustments described (Kiessling et l., 1995; Kiessling et l., 23). The HPV cnnultion hs been described in detil elsewhere (Elison et l., 26). After t lest 24 h of recovery from surgery, the fish were sedted (.1 g l 1 MS-222) nd force-fed single mel of 1% of their body mss by intubtion using stiff PVC tube with rounded tip. The diets were ground through.5 mm screen, nd nlysed for dry mtter (EC, 1971b), sh (EC, 1971), crude Tble 1. Dietry ingredient composition nd proximte nlysis Ingredients (% of diet) Fishmel diet Corn gluten diet Fish mel Whet strch Cellulose Corn gluten EU Stndrd fish oil Vitmin Crophyll Pink CWS 1%.3.3 Mono-clcium phosphte DL-Methionine.13 Proximte nlysis (%) Dry mtter Ash 8 6 Ft Protein (N 6.25) Strch Non-strch crbohydrtes protein (EC, 1993), crude ft (EC, 1998), strch (McClery et l., 1994), non-strch polyscchrides (Lee et l., 1992) nd mino cid composition (EC, 1998). The experimentl design involved two diets (six fish were tested on ech diet), with diet 1 contining fish mel s the only protein source nd diet 2 contining 2% corn gluten s prtil replcement for fish mel. Nevertheless, this djustment produced only very smll differences in the totl nd individul dietry AAs between the two diets (Tbles 1 nd 2). Perhps s result, no significnt effect (P>.5), or ny tendencies (P>.15) were found between the diets for the resulting plsm free AA profiles. Therefore, plsm free AA dt for the two diets were pooled for the nlysis presented here. Blood smples (.4.5 ml) were tken from both cnnule immeditely prior to force-feeding ( h), nd t 3, 6, 12, 24 nd 48 h postprndilly. Whole blood ws centrifuged t 5 g for 5 min, nd plsm ws removed nd immeditely frozen t 2 C prior to storge within hours t 8 C until nlysis. The sttus of the fish ws monitored visully nd with mesurements of hemtocrit nd leucocrit from the blood smples. Plsm levels of lnine minotrnsferse (ALAT; heptocyte specific) nd sprtte minotrnsferse (ASAT; crdic, heptic but lso generl tissue unspecific) were nlysed with stndrdized methods for mesurement of enzymes ccording to the Interntionl Federtion of Clinicl Chemistry (Bergmeyer et l., 1986; Bergmeyer et l., 1986b) on Konelb 3 nlyser using kit no nd for ALAT nd ASAT (Thermo Electron Corp., Vnt, Finlnd), respectively, s indictors of liver dmge (Tble 3). The concentrtions of free AA in plsm smples were nlysed by ion exchnge chromtogrphy on lithium high performnce column (Biochrom Ltd, Cmbridge, UK) in n

3 Postprndil chnges in plsm free AA in HPV nd DA 4887 Tble 2. Dietry mino cid composition Fishmel Corn gluten Fishmel Corn gluten Essentil mino cids diet diet Non-essentil mino cids diet diet Arginine Alnine Histidine Asprtic cid Isoleucine Cysteine Leucine Glutmic cid Lysine Glysine Methionine Hydroxyproline.8.56 Phenyllnine Proline Threonine Serine Tryptophn Tyrosine Vline Turine b Sum EAAs Sum totl c Wter-corrected mino cid; g 1 g 1 crude protein, unless otherwise stted. b Turine; g kg 1 of feed. c Sum of ll AAs, excluding turine. EAAs, essentil mino cids. utomted mino cid nlyser (Biochrom 3, Biochrom Ltd), using lithium-bsed eluents nd post-column derivtiztion with ninhydrin (Physiologicl Fluid Chemicl Kit, Biochrom Ltd). Dt were nlysed ginst externl stndrds (Sigm mino cid stndrd solutions: cidics, neutrls nd bsics, supplemented with glutmine, tryptophn nd S-2-minoethyl- 1-cysteine; ll purchsed from Sigm Chemicl, St. Louis, MO, USA) using the Chromeleon Chromtogrphy Mngement Softwre (Dionex Ltd, Surrey, UK). Plsm (8 l) ws deproteinized by mixing with 8 l of 35% sulfoslicylic cid solution. The mixture ws incubted t 4 C for 2 min nd centrifuged t 16 g for 15 min (Biofuge Fresco, Hereus Instruments, Kendro Lbortory Products GmbH, Hnu, Germny). Of the superntnts, 6 l were diluted with 6 l.2 mol l 1 lithium citrte loding buffer, ph 2.2 (Biochrom Ltd) nd micro-filtrted (.2 m Sprtn membrne filter, Schleicher & Schuell, Dssel, Germny) prior to injection (3 l). Some superntnts were stored t 8 C until nlysis. S-2-minoethyl-1-cysteine ws used s n internl stndrd. Dt were nlysed sttisticlly using the Sttisticl Anlysis System for Windows, Version 8.2 (SAS, 22). The effects of the min vribles, i.e. smple time nd vessel, were tested by min fctoril model (GLM procedure for unblnced dt). Fish ws included s discrete vrible. Groups were compred by the d hoc vrince test (F-test) using the lestsqures mens procedure when significnt effects were found in the min model. All dt were tested for normlity by norml probbility plot (proc univrite plot). P<.5 ws considered to be sttisticlly significnt. Results There were no visul signs of ill helth or infection of the wound nd leucocrit remined norml during the 48 h Tble 3. Alnine minotrnsferse nd sprtte minotrnsferse chnges* in blood plsm collected t (before feeding) nd postprndilly t 3, 6, 12, 24 nd 48 h from free-swimming rinbow trout Postprndil ALAT* ASAT* time (h) (ls men ± s.e.m.) (ls men ± s.e.m.) b ,b ,b b * Ct l 1 ; conversion fctor, 2 i.u. l 1 =.3 Ct l 1, supplied by the mnufcturer. ALAT, lnine minotrnsferse; ASAT, sprtte minotrnsferse. The vlues re the combined result of synchronic smples from both the heptic portl vein nd dorsl ort (P-vlue for difference between vessel ws.74 for ALAT nd.18 for ASAT) expressed s ls-men using smple time (P-vlue for difference between smple times ws.1 for ALAT nd.8 for ASAT) nd fish (P-vlue for difference between individul fish ws for ALAT nd for ASAT) s discrete vribles in the sttisticl model. The sme lower cse letter denotes non-significnt difference (P>.5, using the F-distribution d-hoc test) between vlues obtined t the different smple times. experimentl period. The minor chnges in both ALAT nd ASAT (Tble 3) re not indictive of mjor tissue dmge. There ws definitive ppernce profile for the free AA in the plsm from both the HPV nd DA (Fig. 1). A postprndil pek of the totl free AA concentrtion occurred t 6 h (DA: 717±369 nmol ml 1 nd HPV: 9999±572 nmol ml 1 ). For most individul free AAs, the plsm concentrtions chnged

4 4888 A. Krlsson nd others Totl AA (nmol ml 1 ) c*** b*** c b,c**** b b b b,c*** Time postprndil (h) Fig. 1. Totl free mino cid (AA) levels in blood plsm (nmol ml 1 ; mens ± s.e.m.) collected synchroniclly from the heptic portl vein (HPV; blck squres) nd dorsl ort (DA; white squres) in freeswimming rinbow trout fter single mel. *Sttisticlly significnt difference (P<.5, **P<.1, ***P) between the DA nd HPV vlues t given smple time. Vlues from the sme vessel followed by the sme letter do not differ significntly (P>.5). over time (P<.5), more so with the HPV smples thn the DA smples (Tbles 4 6). All free AAs, with the exceptions of threonine (Tble 4) nd proline (Tble 5) returned to, or below, bseline levels within 48 h. There were three min profiles for the individul free AAs tht chnged postprndilly. All three profiles were chrcterized by n initil increse within 3 h (Fig. 2). Then in the type I profile (Fig. 2A), there ws single pek t 6 h; in the type II profile (Fig. 2B), there ws more grdul rise in concentrtion to pek t 24 h; nd in the type III profile (Fig. 2C), two peks occurred, one t 6 h nd nother t 24 h. The free AAs tht did not chnge significntly were sprtic cid (Tble 5), lph-minodipic cid, bet-lnine, homocystein nd phosphoserine (Tble 6). If there ws no heptic metbolism of the bsorbed free AAs during their first pss through the liver, the rtio of HPV to DA free AA concentrtions would be constnt both over time nd mong individul free AAs, but this ws not the cse. The totl free AA concentrtions were consistently higher (P<.5) in the HPV thn in the DA t 3, 6, 12 nd 24 h (Fig. 1). However, few non-essentil free AAs did not differ between the two smple sites (Tbles 5 nd 6). The highest reltive difference (clculted s percentge difference between DA nd HPV vlues, s given in Tbles 4 6) of n individul AA ws tht of serine (62%) t 24 h (Tble 4), but this occurred well fter the pek uptke of serine t 6 h. The lowest significnt difference ws tht of glutmic cid, which hd pek heptic bsorption of only 25% difference in the 3-h smple (Tble 4). Despite the fct tht the removl of serine nd glutmic cid differed gretly, the liver bsorption of both of these AAs styed elevted throughout the entire 48 h postprndil period. The heptic removl of some of the type I profile AAs (turine, isoleucine, lysine, rginine nd glutmine) styed elevted for considerble period of time, thus the 48 h postprndil AA in % of preprndil control 35 A c* b*** B C b c* b** c b** c* b c,b d** b** b b,c c,b*** b,d b** d*** Time postprndil (h) concentrtion in the DA ws lower thn before feeding (Tbles 4 nd 5). A postprndil pek in the AA metbolites (mmoni nd ure) ws nticipted for the DA blood smples (Fig. 3). However, significnt elevtion in plsm mmoni nd ure in the HPV compred with the DA blood smples ws lso found (Fig. 3). Wheres plsm levels of ure were identicl in the DA nd HPV t the strt of the experiment, they diverged with time, such tht there ws significntly higher plsm concentrtion in the HPV 12 h compred with the DA, before returning to similr levels (Fig. 3). By contrst, the plsm b d*** Fig. 2. Free mino cid (AA) levels in blood plsm (% of h smples; mens ± s.e.m.) collected synchroniclly from the heptic portl vein (HPV; blck squres) nd dorsl ort (DA; white squres) in free-swimming rinbow trout fter single mel. The mjor AA uptke ptterns re represented by the sum of turine, glutmic cid, glutmine, glycine, lnine, lysine, rginine nd serine (type I; A), the sum of vline, cysteine, methionine, isoleucine, leucine, tyrosine nd phenyllnine (type II; B) nd the sum of threonine nd tryptophn (type III; C). *Sttisticlly significnt difference (*P<.5, **P<.1, ***P<.1) between the DA nd HPV vlues t given smple time. Vlues from the sme vessel followed by the sme letter do not differ significntly (P>.5). c c,d

5 Postprndil chnges in plsm free AA in HPV nd DA 4889 Tble 4. Essentil free mino cid concentrtions in blood plsm collected synchronously t h (before feeding) nd postprndilly t 3, 6, 12, 24 nd 48 h, from the heptic portl vein nd dorsl ort in free-swimming rinbow trout fter single mel [Essentil free AA] (nmol ml 1 ) Smple time (h) P vlue Essentil mino cids Cnnul Time Cnnul Arginine DA 84±12 144±14 b,d** 213±15 c*** 14±18 d* 112±16,d** 28±17 e HPV 95±26 266±28 b 382±29 c 271±29 b 257±31 b 49±32 Histidine DA 135±7 17±9 b* 111±9 b 66±11 c* 66±9 c** 62±1c HPV 137±1,b 144±11 141±11 17±11 b,c 124±12,b 82±12 c.13 Isoleucine DA 194±14,c 225±16 * 314±18 b* 329±21 b* 341±19 b** 152±19 c HPV 217±26 327±28 b 422±29 c 474±29 c,d 59±31 d 199±32 Leucine DA 328±38 419±45 * 621±49 b* 721±57 b,c* 821±51 c* 44±53 HPV 373±57 68±62 b 85±63 c 994±64 c,d 196±68 d 468±7,b Lysine DA 35±31,c 437±37,b* 519±4 b** 288±47 c* 266±42 c** 15±44 d HPV 362±5 635±54 b,c 764±59 b 559±57 c 568±6 c 245±61 Methionine DA 56±8 96±9 b** 154±1 c*** 177±12 c,d* 195±11 d** 76±11,b HPV 61±13 152±14 b 228±15 c 245±15 c,d 273±16 d 92±16 Phenyllnine DA 84±6 95±7 ** 129±8 b*** 133±1 b*** 157±8 c*** 85±9 HPV 9±12 143±13 b 182±13 c 196±13 c 238±14 d 16±14 Threonine DA 226±19,e 278±23,c* 394±25 b,d,e* 324±29 c,d** 365±26 d** 288±27 e HPV 234±28 43±31 b 533±32 c 496±32 c 562±34 c 346±35 b Tryptophn DA 2±1,b 21±1,b 22±2 * 17±2 b,c** 23±2 *** 15±2 c.22 HPV 19±2 25±2 b 28±2 b 27±2 b 37±2 c 19±2 Vline DA 419±26,c 468±31 * 642±39 b 647±39 b* 688±35 b* 35±36 c HPV 449±44 639±48 b 86±45 c 889±5 c 926±53 c 423±54 No. of smples DA HPV Vlues re mens ± s.e.m. HPV, heptic portl vein; DA, dorsl ort. Asterisks indicte sttisticlly significnt difference (*P<.5, **P<.1, ***P) between the DA nd HPV vlues t given smple time. Vlues from the sme vessel followed by the sme letter do not differ significntly (P>.5). mmoni levels were lwys significntly higher in the HPV thn in the DA. This difference ws bout twofold t the strt nd end of the experiment, but incresed to over fourfold for most of the postprndil period (6 24 h; Fig. 3) when the free AA uptke ws occurring (Fig. 2). Discussion Experimentl considertions The dorsl ortic rtery cnnultion technique in fish hs become stndrd procedure for short-term blood smpling in lbortory experiments since its introduction more thn hlf century go (see Soivio et l., 1975). Since its estblishment s method for long-term nutrition studies in the mid-199s (Kiessling et l., 1995), it hs been stndrdized, well chrcterized for its physiologicl components nd cknowledged s yielding representtive dt of fish in norml physiologicl stte (B. Djordjevic, T. Kristensen, Ø. Øverli, B. O. Rosselnd nd A. Kiessling, mnuscript submitted for publiction). Fish with DA cnnultion seem to recover their physiologicl nd nutritionl sttus within 24 h post-surgery (Kiessling et l., 1995; Kiessling et l., 23) (B. Djordjevic, T. Kristensen, Ø. Øverli, B. O. Rosselnd nd A. Kiessling, mnuscript submitted for publiction). In contrst to the DA cnnultion technique, HPV cnnultion in fish is more difficult nd intrusive (McLen nd Ash, 1989), but the recovery time is similr, with blood cortisol, glucose, ion, ph, CO 2 nd hemtocrit vlues being restored during the first 24 h (Elison et l., 27). We were plesed to observe norml hemtocrit, leucocrit, nd stble ALAT nd ASAT vlues for the durtion of our 48-h experiments, s well s cler evidence of digestion, nutrient bsorption from the intestine, nd AA metbolism by the liver with the stged, double cnnultion technique used here. The present study clerly shows tht plsm free AA mesurements in the HPV provide much greter resolution of

6 489 A. Krlsson nd others Tble 5. Non-essentil free mino cid concentrtions in blood plsm collected synchronously t h (before feeding), nd postprndilly t 3, 6, 12, 24 nd 48 h, from the heptic portl vein nd dorsl ort in free-swimming rinbow trout fter single mel [Non-essentil free AA] (nmol ml 1 ) Smple time (h) P vlue Non-essentil mino cids Cnnul Time Cnnul Alnine DA 628±57,c 865±67 b** 114±73 b* 464±86 c* 475±76 c* 544±79 c HPV 629± ±92 b 1387±95 b 89±96 c,d 929±12 c 654±14,d Asprtic cid DA 8±,c 8±1,c 9±1 7±1 b* 7±1 b,c 8±1,b.452 HPV 9±1,b 1±1 1±1 9±1,b 8±1 b 9±1,b.1392 Cysteine DA 8± 9± 1±1 b 9±1,b** 1±1 b* 7±1 c.3 HPV 8±1,b 1±1 12±1 b,c 13±1 c,d 14±1 d 7±1 Glutmic cid DA 28±3,d 35±3 49±3 b 34±4,c 29±3,d 22±4 d HPV 3±4 47±4 b,c 56±4 b 44±4 c,d 35±4,d 24±4,d Glycine DA 413±33,c 55±39 * 669±42 b 411±5,c* 353±44 c* 312±46 c HPV 45±46 732±5 b,d 867±51 c 69±52 d 629±55 d 395±56 Hydroxylysine DA 4±1 6±1,c 12±1 b 8±2 b,c* 8±1 c 3±2.3 HPV 3±1 9±1 b,d 14±1 c 15±2 c 12±2 c,d 4±2 Proline DA 45±16 115±19 b** 181±2 c*** 141±24 b,c** 157±21 b,c** 142±22 b,c HPV 49±26 25±28 b 34±29 c 278±3 b,c 298±31 c 161±32 b Serine DA 15±14,e 16±17 b,d*** 243±18 c*** 162±21 d*** 132±19 d,e*** 71±19,f HPV 113±33 327±35 b,d 496±36 c 376±37 d 345±39 d 123±4 Turine DA 292±41,d 417±49 655±53 b 393±62,c 183±55 d,e 136±58 e HPV 332±67 621±73 b,d 826±75 c 551±76 d 193±8 155±82 Tyrosine DA 65±9 87±11 * 128±12 b 136±14 b,c 165±13 c** 57±13 HPV 66±11 124±12 b 162±12 c 178±13 c 225±13 d 72± Vlues re mens ± s.e.m. Asterisks indicte sttisticlly significnt difference (*P<.5, **P<.1, ***P) between the DA nd HPV vlues t given smple time. Vlues from the sme vessel followed by the sme letter do not differ significntly (P>.5). the uptke profiles thn do DA mesurements, presumbly becuse of heptic metbolism. The free AA concentrtions reported here for DA smples re comprble to erlier studies where only the DA ws cnnulted (Ok et l., 21; Sunde et l., 23). In generl, our dt lso gree with the erlier study Ammoni in plsm (nmol ml 1 ) c*** c*** c*** b*** c* ** b,c 1 b b,c * 1 b c b 5 b b Time postprndil (h) Ure in plsm (nmol ml 1 ) of Ash et l. (Ash et l., 1989), who mesured the postprndil concentrtions of free AA in both the DA nd HPV of lightly nesthetized rinbow trout, but only t 3 h. They found much smller increse from the bse line (48 h strved fish) t 3 h nd smller differences between the DA nd HPV smples compred with the present study. However, beyond generl comprison with this erlier study, detiled comprisons re complicted by the reporting of blood rther thn plsm concentrtions nd by mesuring bseline nd postprndil vlues in different sets of fish. Also, Ash et l. occluded mjor route from intestine to HPV by cnnulting the intestinl vein, Fig. 3. Postprndil concentrtions (nmol ml 1 ; men ± s.e.m.) of mmoni nd ure in blood plsm collected synchroniclly from the heptic portl vein (HVP) nd dorsl ort (DA) in free-swimming rinbow trout fter single mel [mmoni in HPV (blck squres), mmoni in DA (white squres), ure in HVP (blck tringles) nd ure in DA (white tringles)]. *Sttisticlly significnt difference (P<.5, **P<.1, ***P<.1) between the DA nd HPV vlues t given smple time for either mmoni or ure. Vlues from the sme vessel followed by the sme letter do not differ significntly (P>.5).

7 Postprndil chnges in plsm free AA in HPV nd DA 4891 Tble 6. Endogen (not present in diet) non-essentil free mino cid concentrtions in blood plsm collected synchronously t h (before feeding), nd postprndilly t 3, 6, 12, 24 nd 48 h, from the heptic portl vein nd dorsl ort in free-swimming rinbow trout fter single mel Endogenously Endogen [non-essentil free AA] (nmol ml 1 ) formed non-essentil Smple time (h) P vlue mino cids Cnnul Time Cnnul 1-Methylhistidine DA 9±3,d 2±3 b* 39±4 c* 31±4 c 16±4,b 4±4 d HPV 7±4 36±5 b,c 55±5 c 48±5 c 22±5 d 7±5,d lph-aminodipic DA 17±1,b 14±2,b 18±2 13±2 b 15±2,b 16±2,b.1841 cid HPV 15±1 18±1 18±1 16±1 15±1 14± lph-amino-n- DA 29±2 26±2 32±2,b 26±3 32±3,b 37±3 b.171 butyric cid HPV 3±2,c 33±3,b,c 38±3 b,d 37±3,b,d 42±3 c,e 42±3 d,e.177 Asprgine DA 129±13,c,d 145±15,c** 29±17 b*** 15±19 c** 145±17 c*** 94±18 d.3 HPV 134±2 234±22 b 32±22 c 269±22 b,c 299±24 c 123±24 bet-alnine DA 31±3 26±4,b 34±4,c 27±5,b* 16±5 b 22±5,b.51 HPV 22±5 36±5 b,c 43±5 b 44±5 b 21±6 22±6,c.9 Citrulline DA 18±1,c 2±1 28±1 b 3±2 b 28±2 b 14±2 c HPV 16±2 24±2 b,d 3±2 c,d 35±2 c 29±2 d 15±2 gmm-amino- DA 11±1,b 9±1 12±1 b 11±1,b 12±1 b 1±1,b.83 butyric cid HPV 1±1 12±1,c 14±1 b 15±1 b 13±1 b,c 1±1.5 Glutmine DA 271±23 281±27 * 393±29 b* 278±34,c* 252±3,c** 158±32 d HPV 281±3 415±33 b,d 53±34 c 442±34 d 43±36 d 22±37 Homocystine DA 23±1 19±2 22±2 18±2 2±2 18± HPV 21±1,b 21±2,b 22±2,b 24±2 23±2,b 19±2 b.2394 Hydroxyproline DA 38±4 43±5 76±5 b 7±6 b,c* 59±5 c,d 47±6,d HPV 35±7 62±8 b,d 98±8 c,d 112±8 c 83±9 d 55±9,b Ornitine DA 51±3 57±4 74±4 b 47±5,c,d 47±5,c 34±5 d HPV 53±4,c 74±5 b,c 83±5 b 68±5 c 61±5 c 44±5 Phosphoserine DA 12±1 1±2 11±2 1±2 1±2 11±2.937 HPV 1±2 11±2 13±2 13±2 14±2 13± Vlues re mens ± s.e.m. Asterisks indicte sttisticlly significnt difference (*P<.5, **P<.1, ***P) between the DA nd HPV vlues t given smple time. Vlues from the sme vessel followed by the sme letter do not differ significntly (P>.5) wheres we minimized vessel occlusion by cnnulting smller side vein off the min dorsl nd ventrl intestinl veins (Ash et l., 1989). Since the concentrtion of free AAs in the HPV is probbly combintion of the rtes of intestinl blood flow nd AA uptke, ltertions to intestinl blood flow could lter free AA concentrtions. Therefore, comprisons with nesthetized fish re compromised becuse nesthetic procedures nd hndling reduce gut blood flow (Thorrensen et l., 1993; Elison et l., 27). Sedtives could lso ffect AA uptke rtes even though Kolnczyk et l. did not find ny significnt chnges in liver enzyme ctivity fter exposure to MS-222 (Kolnczyk et l., 23). Ok et l. found tht postprndil plsm free AA concentrtions in the DA peked t 4 h, except for glycine (Ok et l., 21). Here, the totl free AA concentrtion peked t 6 h, time frme tht grees with the results reported fter seril smpling of slughtered fish (see Sunde et l., 23; Espe et l., 1993). This slight dely in the pek of free AA compred with those found by Ok et l. my be relted to the wrmer temperture (~17 C) used in the experiments of Ok et l. compred with the two subsequent studies (~1 C), lthough n effect of dietry differences cnnot be excluded. Ok et l. used combintion of crystlline mino cids nd csein nd geltine (Ok et l., 21), nd crystlline AAs re known to be bsorbed quickly (Cowey nd Wlton, 1988). A more rpid uptke should result in higher nd shrper peks of plsm free AAs. The three different uptke ptterns of free AAs observed in the present study re difficult to confirm. Neither Ash et l. (Ash et l., 1989) nor Sunde et l. (Sunde et l., 23) used repeted smpling, wheres Ok et l. used n rtificil diet, possibly compressing digestion (Ok et l., 21). Lyndon et l.

8 4892 A. Krlsson nd others found both single nd double peks in uptke profiles for cod (Lyndon et l., 1993), but their dt show gret divergence for the sme free AAs between the two smple sites, suggesting mbiguous results due to serilly slughtering group of fish to obtin blood smples from the HPV nd by crdic puncture. Espe et l. lso used seril slughter with Atlntic slmon (Slmo slr) nd reported three uptke profiles using cudl puncture to obtin blood (Espe et l., 1993). A mixed rtery vein smple mkes direct comprisons to the present work difficult. In the present work, it ws cler tht free AA uptke ws well underwy by 3 h, despite the gvge method, nd tht the mximum uptke rtes, s judged by the HPV concentrtions, occurred between 6 nd 24 h, depending on the individul AA. Free mino cid uptke profiles We observed three different free AA uptke profiles. Different AAs re digested nd bsorbed by different mechnisms, grossly clssified s ctive nd pssive crriers nd chnnel-medited diffusion. In ddition, di- nd tripeptides re bsorbed independently. In fct, s much s 7 85% of ll luminl AAs my be bsorbed from the digest into enterocytes in the form of smll peptides (Krehbiel nd Mtthews, 23). However, fter bsorption into the enterocytes, the peptides re further hydrolysed intrcellulrly. As result, most of the AAs ppering the heptic portl vein re free AAs (Krehbiel nd Mtthews, 23). Such differences in bsorption mechnisms could explin the three profiles observed here. With the exception of cysteine, ll the AAs with uptke profiles 2 nd 3 (Fig. 2B,C) re known to be tken up by the mmmlin gut vi ctive trnsport mechnisms. Similr ctive trnsport mechnisms my exist in fish, lthough they re not yet identified (Smith, 1989; Hlver nd Hrdy, 22). Thus, if the number of trnsporters is limited, the uptke profile will hve longer durtion. By contrst, AAs bsorbed vi pssive crriers nd diffusion will be concentrtion dependent, nd smll peptides re known to be bsorbed more rpidly (Steinhrdt nd Adibi, 1986; Mtthews, 2; Bogé et l., 22; Dbrowski et l., 25). An lterntive explntion for the plsm free AA profiles (Fig. 2A C) is regionl bsorption of specific AAs, with the ones displying slower pek being bsorbed in more distl prts of the intestine. This is less plusible explntion becuse severl studies indicte tht the mjority of AA nd peptide bsorption occurs in the proximl- to mid-intestine (Webb Jr, 199; Mtthews, 2). The AAs for which there were two peks (tryptophn nd threonine, Fig. 2C) could be n exception in tht the second pek could prtly reflect rebsorption of AAs from proteolytic enzymes contining significnt mounts of tryptophn nd threonine in the distl intestine. Also, Umezw et l. (Umezw et l., 1985) showed tht co-dministrtion of leucine nd tryptophn my dely the tryptophn bsorption. Free AA uptke efficiency nd metbolism Finding similr pre- nd post-digestion plsm free AA concentrtions ws n encourging qulity control for the experiments nd therefore we feel confident bout discussing the rtios of free AA in HPV nd DA smples. We expected the HPV concentrtion to be elevted over the DA concentrtion becuse blood returning to the sinus venosus from the heptic circultion is diluted by other systemic venous return in direct proportion to the reltive proportion of heptic blood flow. Bsed on vilble literture (McLen nd Ash, 1989; Thorrensen et l., 1993; Elison, 26) nd personl observtions, roughly 3% of crdic output is chnnelled vi the HPV in resting nd unfed fish nd hs been estimted s 12.8 ml min 1 kg 1 fish (McLen nd Ash, 1989). Gut blood flow increses by 6 1% fter mel nd pproches 5% of crdic output (Thorrensen et l., 1993; Elison, 26). Hd we mesured these reltive flows in the present experiment, we could hve directly clculted uptke efficiency s ([AA HPV ] [AA DA ]) blood flow)/totl intke of AA, nd then estimted liver metbolism of individul free AAs from their respective DA nd HPV concentrtions. Despite the lck of blood flow dt, we still found evidence of heptic nd systemic metbolism of free AA becuse the concentrtion rtio vried mong AAs nd over time. If there ws no heptic metbolism of the bsorbed free AAs during their first pss through the liver, the difference between the HPV nd DA free AA concentrtions would reflect simple dilution by systemic venous return nd, bsed on the bove blood flow distributions, 5% dilution is resonble dilution for the postprndil stte. Such dilution cn explin the HPV DA difference for severl (e.g. glutmic cid nd turine; Tbles 4 6), but not ll free AAs. These free AAs lso hve prllel curves throughout the ctive bsorption phse (3 24 h smples). A complicting fctor in this estimte is the possibility of n ccumultion of AAs in the plsm s neither the heptic or systemic tissues completely remove ll AA before the blood is returned to either the intestine or the systemic vsculture. In cses where the HPV curve shows shrper inclintion before the mximum pek, compred to the DA curve, we ssume tht heptic bsorption domintes (e.g. vline, cysteine, tryptofn, threonine, serine; Tbles 4 nd 5). However, with mjor removl of AAs by the systemic tissues, such s muscle, the dilution effect will become more prominent, resulting in n incresed HPV DA difference. But such sitution should be signified by reduction in concentrtion in the DA rther thn by n increse in the concentrtion in the HPV, especilly fter the bsorption pek, s seen for, e.g. histidine, isoleucine, lysine, rginine, glycine nd glutmine (Tbles 4 nd 5). An dditionl novel discovery here is the possibility of intestinl metbolism of AAs before they rech the liver. This conclusion is supported by the fct tht not only ws the mmoni level higher in the HPV thn the DA, but tht the difference between the two blood smpling sites incresed during free AA bsorption such tht the plsm mmoni levels were three times higher in the HPV thn the DA. Thus, there is cler role for the liver in protecting the rest of the fish from elevted nd perhps even toxic levels of mmoni in the

9 Postprndil chnges in plsm free AA in HPV nd DA 4893 generl circultion. There ws different signture for plsm ure, with significnt difference only existing t 12 h. These results suggest tht there is significnt demintion of AAs, nd to lesser extent ure formtion, before blood from the intestinl mucos reches the liver vi the HPV. Utilising the sme clcultion s given bove, the mount of deminted AAs could be estimted, yielding informtion on the effect of diet ltertion on AA s substrte for fuelling the digestion processes, especilly if lbelled substnces were used. At the sme time, the mjority of mmoni [min metbolite of AA demintion in fish (Hlver nd Hrdy, 22)] is excreted cross the gills just before the DA smple site, thereby yielding bseline vlue for the plsm entering the intestine. To wht degree mmoni nd ure might be generted in the lumen of the intestine during digestion, nd pss directly into the heptic portl blood is uncler. However, Mommsen et l. discovered high ctivity of glutmine synthetse in tilpi gstrointestinl trct nd hve suggested its role is in mmoni detoxifiction (Mommsen et l., 23). Our dt concur with the need for such role, but suggest tht if present in Atlntic slmon, the detoxifiction role is prtil nd heptic metbolism in combintion with gill excretion re dditionl components of such protective detoxifiction system. In conclusion, the present work shows tht double cnnultion in rinbow trout is fesible tool for physiologicl studies of nutrition in fish nd cn be used to ccurtely trck uptke profiles of individul free AAs nd provide much deeper pprecition of underlying mechnisms compred with informtion provided by single DA cnnultion. In ddition, we provide cler evidence in support of the hypothesis tht there is heptic metbolism of certin mino cids during their first pss through the liver fter bsorption by the intestine. Furthermore, by combining these types of studies with mesurements of blood flow, it should be possible to ccurtely estimte tissue ssimiltion nd nutrient ctbolism of specific nutrient during digestion. This study ws finnced through the Norwegin Reserch Council, Centre of Excellence, Aquculture Protein Centre (APC). Additionl funds, in kind, ws lso provided nd hereby cknowledged by the Deprtment of Animl nd Aquculturl Sciences, nd by the Deprtment of Mthemtics nd Technology, Norwegin University of Life Sciences, Norwy, to A.K. The Nturl Sciences nd Engineering Reserch Council Cnd provided funding for A.P.F. References Ash, R., McLen, E. nd Westcott, P. A. B. (1989). Arterio-portl differences nd net ppernce of mino cids in heptic portl vein blood of the trout (Slmo girdneri). In Aquculture A biotechnology in progress (ed. N. De Puw, E. Jspers, H. Ackefors, N. Wilkins), pp Europen Aquculture Society, Bredene, Belgium. Bergmeyer, H. U., Horder, M. nd Rey, J. (1986). 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