14

Effect of glucose and branched chain amino aud (BCAA) infusion on onset of liver regeneration and plasma amino acid pattern in partially hepatectomized rats

Immrdiatci> after 70% bsp;l:ecromy, rats were infused via the jugular van with one of the following solutions: saline, 3% or 8% gh~osc solution, 3% BCAA solution, 8% Nutramin (17% BCAA) or 8% Nutramin enriched with BCAA (4?% BCAA). Rats WC-~killed l&21,24 and 30 h after partial hepatectomy. The courseof liver regeneration was assessedfrom incorporation of labeled thymidine into DNA and from hepatocyte mitotic activity. After partial hepatectamy a decrease of glycemia and increase of plasma phenylalaninr concentrations were observed. Glucose infusion had a pronounced inhibiting effect on the onset of liver regeneration. The decreasedincorporation of labeled thymidine into DNA and the decreased hepatocyte mitotic activity in glucose-infusedrats we:e associatedwith higher plasma concentrations of tyrasine and phenylalanine. Parenteral administration of 3% BCAA solution and 8% Nulramin enriched with BCAA had a favourpble eifect on the onset of liver regeneration after partial hepatectomy compared with giucase- and Nutramin-infused groups. In BCAA-infused rats elevated lsvels of ECAA and decreasedconcentrationsof tyrasine and phenylalanine were noted

Survival aher hepatocelhdar injury and necrosis may depend on the ability of the remaining hepatocytesto regenerate and restore an adequate population of functmning cells. A classicalmodel which enables us to study the rate of liver tegaention is the model of regenerating the liver after partial hepatectomy (PH). In clinical apphcation we can encounter panlal hepatectomy in casesof cyst, neoplasla or hepatic resection due to trauma. In the rat. in which Ihis processis srudxd most ofren, 65-70% ot the liver is usually removed (1.2). Liver regeneration after PH in the rat manifests itself from approximately the 14th h after operation with a gredual increase in DNA synlhesir. thr maximum occurring around the 24th h (2).

Approx. 6 h later (30 h past-operaban). the number of hepatocyte mitosesbegins to increase(3). Adequate nutritional suppon plays an importzz: ioL in facilitating liver regeneration after PH (4,5), representing a physiological treatment which simultaneously satisfies the energy demandsof the organism. However, much has yet to be discoveredconcerning which nutritional factors arc critical for liver regeneration. Since it is well-known that marked hypoglycemia follows PH (6), it is commonly accepted that carbohydralcs are a necessarycomponent in nutrition. On the other hand, resultsof studiesconcerning the metabolism of amino acids and their plasma levels after PH are not as clear (7.8). Therefore alterations of

CLUCOSE,

EC**

AND

w/m

REFENER.m,ON

15

amino acid metabolism occunmg after PH should be better defined so that they can be adjusted to meet the need of the organism. In a clinical aPplication of amino acid analysis, Fischer and Baldessanm tr) reported that the mo?zrratio of ptasma BCAA (valine. leucina, is&wine) and aromatic amino acids (AAA. tyrosine. phmylalanine) (BCAAI AAA) decreased as the severity of hepatic parencbymal

MJil - which IS a 3% BCAA. Solution containing (per line) 10.0 g valme, 12.77 g leucinc and 1.23 g isoleucine.

damage increased. Subsequently, other investigators have reported that BCAA administration to patients with severeliver damage bad a favourable effect on the coneclion of negative nitrogen balance and a preventive effect in the dwslopmenl nf hepatic encephalopathy (10.11). In this work we attempted to show how the infusion of gla-

23 Energy content is approx. 0.9 MUI. After termina:ion of the infusion the rats were given only water until they were killed by decapitation 18. 21. 24 or 30 h after PH. The cowse of liver regeneration WBE rvaluated by observingthe changesin specific DNA activitv after IR. 21.24 and M h oost-PH. One hour before killing,‘“C-thymidine was inlected to the rats via a catheter in a dose of 22.2. IdJ Bqlkg (specific activity 0.8l~IoX Bqlmol). Liver samples for measuring specific actwity were worked ou! according to Short et al. (12). Radiaactwity of sampleswas measured by msansof a Delta 300 (Nuclear Chicago) scintillation radioactivity computer. DNA content in liver sampleswas determined

CMOand amino acid solutions with different BCAA contents influence the onset of liver regeneration and amino acid plasma levels in panially hepateaombed rats.

Experiments were carried out on 350 Wistar laboratory rats, six to nine in a group. weighing 180-200 g each. In the first stage of the study we attempted to analyze changesin glycemia and individual amino acid concentrations in plasma following 65-70% hepatectomy. To exclude nutritional effects on aminoacidemia, the rats were only given water doting the 12 h before the experiment, and then PH was cartled out under light diethyl ether narcosis (1). The control sample consistedcf two groups of rats. In one, sham.operations were performed after I2 h of fasting, and in another there was fasting only for 12 h G:bzt ar.y oprarion. Four, 8 or 24 b after operating, blood was taken from the aorta bifurcation. Ammo acid concentrations in the plasma were determined after deproteinhation of the samplesby sulphosalicylic acid using an automatic amino acid analyzer (T339, Miktwtechns). Giumse concentrations in the blood plasma were determined enzymatically. In the second part of out study 65-70% hepatectomy was carried out and a catheter was inserted into the vena jugularis dextra. Immediately after the operation, 5 ml of one of the following tested solutions was administerea in a 4-h long infusjon. Solutions tested: (a) saline solution (Sal); (b) 3% @rcose (G3) - energy content 0.5 MJII; (c) 8% glucose(GS) - eoergy content 1.4 MJil; (d) 8% Nutramin Spofa (Nu) - see Table I. BCAA content is 17% of all amino acids, energy content represents 1.4 MJ,l; (e) 3% Nutramin VLI Spofa (VLI) - energy content 0.5

VLl i: assd as 8 suoo:ementto commercial amino acid aulutions accordmgto the needsof the patient; [‘: 7% :iu:ra;lli.~ ixl;i;i& nirt 3% Nmramin VLI (Nu + “t-1) so that the BCAA share formed approx. 47% of a,, amino acids. Tbis ratio was reachedby mixing Nu with VL! in a ratio of

by the diphenylamine reaction (13). The mitotxc activity of hepatocyteswas read from 7gm thick paraffin sectionsof liver tissue stained with tnema-

toxglin and cosin The mitotic index equalled the number of mitow per lOOn hrpatocyte nuclei (3wO in each rat sere evaluated). Plasma glucoseand amino ?cid concsntrationswere estimated 24 h after partial hepatectomy in saline, glucose and VU infusedgroups. The means f SE. of six to nine animals are indicated. Significance ot dntferencesoi rbe mean, ~a; cbxked using f-lest and Student‘sr-test.

Parunt hepatectomy was followed by a significant decrease in glycemia. while its increase was noted after the shamaperation (Fig. 1). Plasma BCA4 showed little or no variation over the periods studied. However, after PH a significant increase in AAA was found when compared with sham-operated groups (Table 2). Due to the elevated levels of AAA the plasma BCAAiAA.4 ratio in partially hepatectomizrd rats decreased, white after the sham-operation it increased(Fig. 2). The effect of the infusion of each of the tested solutions on the onset of hver regeneration is demonstrated in Table 3 and Fig. 3. Eighteen and 21 h after PH, i.e., in the period of the increase in DNA synthesis(2). we observed significxtly !ower values of specific DNA activity in rats to wham glucoseor Nu were infused compared with Sal or VLI infused rats. After infusion of an amino acid solution enriched wit WAA (Nu + VLI), we observed significantly higher valriesof specific DNA activity @ < 0.05) 18 h after PH compared with rats to whom a solution of

‘I 4

GLUCOSE.

BCAA AND LIVER REGENERATION

TABLE 3

aminoacids without BCAAenrichment (No) was infused. The specific DNA activity 24 and 30 h after PH did not differ among the individual experimental groups (Table 3). The valuesofmitotic actiwty ofbrpatofytes 24 and 30 b after PH (Fig. 3) correlated mostly with the values of specific DNA activity after 18 and 7.1 b. In rats infused with glucose or No the values of mitotic activity were lower when compared with the remaining experimental groups. No difference was found in plasma glucose concentrations among control and experimental groups(Fig. 4). After VLI infusion a significantlv higher level of valine (p < 0.01) and decreased concenrrations of tyrosine @ c 0.001) and phenylalanine (p < 0.01) were found con,. pared with the saline-infused group. On the other hand, in GS-infused rats the plasma eromatic amino acid levels were hi&her (:yrosine: p C U.05) than in saline-infused rats (Fag. 5). 15 20f

T

r

In rats with PH infused with Sal or G3. a significantly decreased BCAA/AAA ratio was bond. On the contrary, in the VLI infused group an increase of BCAA/ AAA ratio war noted, causedby a higher level of valine and decreased plasma ct~ncentration~of tyrosine and phenylalanine (Fig. 6). Fig. 7 shows the infusion influence of !he tested solutions on the plasma levels of some other amino acids. Af. ter G3 infosion significantly higher levels of alanine, proline and glutamate @ < 0.05) were found ihan in the saline-infused grasp. VLl infusion led to a decreasein methionine towards the control value @ c 0.05) when compared with the Sal-infused group.

Discussion The decreaseof glycemia observed after PH is caused by removing mare than 213 of the liver gtycogencontent

M. HOLECEK

IX

et al.

and tyrosine concentrations in blood plasma increased and this led to a decreasein BCAA/AAA ratio which is of prognostic importance in clinical practice (18). The specific activity of DNA and the hepatocyte mitotic

and by the nonnpecific stressof the operation (6.14). A number of studieshave demonstrated that glucoseutiliza-

activity of saline-infused rats did not differ compared with non-infused PH rats which were only cannulated (data not shown). This means that the saline group is a suitable controt group. We found that none of the groupsinfused with gtucoseand/or amino acid mixtures, exceeded the values of DNA specific activity of the saline group at 18 and 21 h after PH. This indicates that an infusion of a glucose and complete amino acid mixture given within the first 4 h after PH suppressesthe onset of liver regeneration after PH. It can he presumed that the inhibitory effect of glucose infusion on liver regeneration, also observed by other authors (19-Z), is caused by a metabolic block which re. dues the ability of the liver to utilize glucose in an aerobic

ttw F-y the remaming livex tissue after PH is decreased and gluconeogenesisis increased (15.16). These changes gmdually lead to rrstomt~on of glycemia to the pre-operatiw Y.I”CS. To clucidats the mechanismand importance of arncentration changesin the individual amino acids after PH is

way. Fatty acids, rather than carbohydrates, are the preferred substratefor energy-dependent metabolism in the liver remnant soon aher hepatectomy (22). This explanation is consistent with our recent finding that, compared with saline and glucose infusion, lipid emulsion infusion has a stimulatory effect on the onset of liver regeneration

not easy. IIese

(21). Since glucose infusion is used in human surgery for hypoglycemia correction after partial liver resection (23), we believe that its inhibitory effect on the onsetof liverw generation should be taken into consideration. Moreover. in partially hepotectomized rats higher levels of tymsine and phenylalanine following glucose infusion indicate the onset of amino acid disturbances which accompany hepatic insufficiency. R&y. there is no doubt that for successfulregeneralion the liver needs amino acids. PH rats on a protein-free diet synthesized less DNA than rats on a normal diet

changesare caused not only by the re-

moval of ?O% of the liver tissue, but also by the stressof the operation. changes in gluconeogenesis, increased turnover of amino acids in the remaining liver associated wi!h the increased rate of urea production, muscle m;ss !typrrcatabolism. and other factors, all of which should ‘5e taken into account. Our main interest were changes in BCAA and aromatic amino acid concentrations in re’adon to the metabolic changes of these amino acids in hep atoceltular injury (10,17,18). Plasma BCAA concentmtions did not change after PH. However, phenylalanine

(24,25). From our results we can surmise that similar to ghxose infusion, the infusion of amino acid solutions, balanced according to the formula of Rose (26,27), retards the onset of liver regeneration following PH. Compared to saline. the administration of BCAA in the form of 3% Nutramin VI.1 did not significantly influence the onset of liver regeneration. The enrichment of the commercial amino acid solution (8% Nutramin) with BCAA partially eliminated the retardation effect of other amino acids on the rate of liver regeneration. In BCAA-infused rats, liver regeneration began earlier than in glucose-or Nutramin-infused rats. This may take on greater significance if extrapolated to man, in whom the measurable onset of livet regeneration does not occur until several days after surgery. A favourabte effect of BCAA infusion on the develop ment of liver :egeneration has also been observed by Ri-

GLUCOSE, BCAA AND LIVER REGENERATlON Sotti et al. (2R) and Saint-Aubert et al. (14). In contrast Kirsch and Saunders(29) noted that following intragastric ndministration of BCh.A, DNA specific activity values were lower thaa with a comPlete amino acid mixture. It may be presumed that the method of admmistration (p.o. orP.e.) andotherfactors. e.g., duration and volumeof infus!on, significantly influence BCAA utilization and the effect on liver regeneration. Twenty-four and 30 h after PH. DNA specific activity rn glucose- and Nutramine-infused rab was higher, a,-

19 of valine exceedsthe muscle’scapactty to metaboltze this substancein these sitoatioos. Schauderet al. (32) found a diminished tolr~ance for valinc end for its corresponding keto acid in Patients with cirrhosis compared to conrro, subjects, hut not for leucine. This suggeststhat nrrhotics have a defect in one or more of ;he metabolic neps after branched-cham keto aad dehydrogenase. It IS probable that similar changesdevelop k4,owing partial hepatectamy, hypotheses which couid ialso explain our results.

a

WC are aware that the different energetic values of the

though not significantly, than in the Saline and BCAA groups, and can be explained by compensatory changes. A similar phenomenon was observedby Rigotti et al. (29) 4d pod 72 h after 75% hrpatartvrny io rats infused aatd kilIing. Since liver regeneration in rats is a rapid process and in our study the tested solutions were only infused for !he first 4 h following surgery. the compensatory changes developedearlier than those describedby Rigoth et al.

tested solutions

In the group which received 3% BCAA solution, oormal levels ot tyrosme, Phenylalanine. methionine. and alanine ‘were achieved. Higher levels of valine and decreased plasma concentrations of tyrosine and phenylalanine in the VL,-infused group led to an increiise in the BCAA/AAA ratio, a sign of improvement in both plasma

DNA specific activity and hcpatocyte mitotic ac:ivily are higher in the YLI-infused group than in the grows recaving a glucoseor complete amino acid mixture in-aion. In addition. the aim of our work was to test the in xncc of individual amino acid so,ufions which are prer~’ tly used for clinical application. h is not simple to explain the mechanismof thL effect of

amino acid Profiles and liver function. These results support Rigotti et al. (28) who found elevated levels of BCAA and decreasedconcentrationsof tyrosine. pheny;alanine, and mcthianine following a BCAA-enriched solutioo infusion in rats with 75% hepatectomy. An interesting finding of passihle clinical importance is rhe high level of valine concentration in VLI-infused rats 24 h after PH. However, it is difficult to explain this finding. Similar increasesin valine concentration, were found by Freund et al. (30) in the plasma, musclesand bvers, of rats follwicg !qxoiilmy dud by Swendseid et al. (31) m the Plasma of Patients who had fasttd, although the same concentration (WV) of valine, ieucine and isoleucine were infused. Valine infusion increasesmuscle Protein synthesis more than other BCAA (30), perhaps since the :;i:!y

rcprerent

a certain

shoncoming

in our

work. The energetic values of the main solutions which wre compared (0.5 Ml/l in the 3% glucose and 3% BC.41?, end !.4 MJfl in the SJ glxose md 6’: N!rramin) are. however, approximately the rar 0. Froa oar resuits it is clear that the energy content of Infused alotions has no effect on the conclusionsthat glucoseand :omp,ete amino acid mixtures have an inhibitory effect !nd that

individual amino acidson the de&opulent of It :?r tissue rsgeneratioo. Priority BCAA utilization by muscletissue has a significant effecr on the metabolic slate of the whole organirm. It should also be kept in mind that an infusion of ai-rino acids affects release of glucagon, insulin and other hormones(33,34). In this situation, for instance,the effect of BCAA on liver regeneration may be mediated by the known synergistic action of insulin and glucagon (35.36).

Acknowledgemen& iv,wy thanks ior he@ in revision of the English 10 Dr. I. CtWxZek and A. Faust.

20 aminn acids in pwtially hcpalectomizcd rats. Bmchim Biophys Ac,a 107:. 173 7: u 8 StrLckcr W. Silz S. Salem A. Ruhcnrtruih-Bsuer G. Metabnlic changerin the serdrnof psrtinlly hcprtcctomized ruts. Horm Mctab Rcr 198u: 12:KW-R. 9 FischerJE. BnldcssariniRI. Falrc ncurotransmittcrr and hepatic bilurc. Lnnccl 1971, ii: 75-80. IO Rossi.Fanelh F. Rigpio 0. Cangiano C. Branchrd-chain wmua ncidr v’i lnctuln~ein the trcatmcnt of hcpatic comz~.Dig Dis Sci 1YRl:27: 929-35. II Watnnabe A. Shiotn T, Okita H. Nagarhima H. Efft*ct of brunched chsio amino arid-enriched nutntlonal produn on the pstncphyriologyof the liver and nutritional state01 patients with liver cirrhuslr Acta Mcd Okaynmn 1983; 37: 321-33. 12 Short J. Zcmcl R. K~ntn J, Lieberman t. Stimularion ofdeoxyrihonucleic acid synthesisin the liver parenchymalcell8 of the intat, mt Nnturc 1969;223: 456-7. 13 Burton K. A study of the condlnnn and mcchanwn of the culorimctnc esnmation of dcanyribonucleicacid. J Biochem 1956: 62: 315-7.3. 14 Saint-Auberr B. AstroC. Andriguctto PC, Ynkoun H. JoycuxA. fnfluencc of nutrition on liver rcgcncrotiou. In: Kleinberger G. Deutsch E. cdr. New AsPcctr of Clinical Nutrition. BaseI: KC+ gcr. 1983:54& 57. 15 Camarga Gluoonecgenesisin liver shces from partially xpatectomized rats. Proc Sot Exp Biol Med 1971; 136:*2-6. 16 Yildirim SI. Paulsen HE. Ouantnalive liver functions after 70% hepntectomy Eur J Clin Invest 1981: I : 469-72. I7 FrschcrJE. FnnovicsJM. Aguirre A. The role of plasma amino acidsin hcpntic cncephalopathy.Surgery1975; 78: 276-90 I8 Morgsn MY. Wilson JP. Sherlock S. P&me ratio of valine. Icucute snd isoleucineto phenylalantneaud tyrorine in liver disease. Gut 197fl; 19: 11~8-73. 19 Bengmark 5. Olrron R. Svsnbore A. The influence of glucose supplyan liver rwatosis and trgeneration rate after hepa~ecloruy.AcrsCinrScmd 1965: 130.216-23. 7” N:z!j ICIF. Lambaue L. Effect of glucose toad an AI-P and DNA synthesisin the rcgcnerating rat liver. Arch Int Physici Biochem 1981;75: 12-3. 2, Hale&k M. $irnek J. D,ffcrent effcc,o‘@cose and fntratipid on the onset of liver regenerxion in the early period after partial hcparectomy!n ihe rat. Exp Palhol 1988;?> 257-M). 22 Nakatani T. Orswa K, Asarm M. Ukikura M. Karniyama Y, Tobe T D,ffcrences in predamrnanrenergy wbstrate in relauon

ACM. Miphonni W-l.

1

partial

to the rcscctcdhepatic masxm the phase:,mmcdiatelyafter hepntcctomy. J LabClin Med lYS1:97: 887-98. 23 Price 18. Schullinger JN. Santulli TV. Major hcpstic resection for neoplssiainchildren. ArchSuig IYRZ; 117: 1139-41. 24 Dsllman PR. Manies EC, Protein deficiency:cootrartinp rffcils au DNA and RN.4 melnbolirm in rat liver. J Nutr 1974; 103: 1311-8. 25 Bllkkcndaat-Lirftinck LF. Kno,i M. Kramer MF. Otter WD. Cell Linetim in the liver of ruts under normal and abnormal dietary conditions. An autoradiographicstudyby meansor HI-tbymidine. Exp Mel Pathot 1977;2b: 164-92. 26 Rose WC. Amino acid requirementsof man. Fed Proc 1945; B: 546-52. 27 RuseWC. Smith LC. Wumaak M, ShoneM. The utiliition of nitrogen of wmnunium salts. urea snd certain other compnmdr in the synthesisal nonessentialamino acids in vivn. J Biol Chcm 1949; 181:M7-16. 28 Rigotli P. Peters JC. Tranberg KO, Fischer JE. Effect of amino acid infusions un liver regeneration after partial hepatectomyin the rat. J ParenterEnter Nutr 1986; 10: 17-20. 29 Kirssh RE, SaundersSJ. Frith KIC. Rawtings R. Wmdbum Y. Thcsffect ofintragastric fccding\uithaminoaEidson liver regeneration after partial hepatectomyin the Am J Clin Nutr 1979: 32: 738-40. 30 Freund HR. James JH. Fischer JE. Niwogen-sparing mccbrz nisms of singly administered brunched-chain amino acids in the injured rat. Surgery 1981;90: 237-43. 31 SwendseidME. Villalolios I, Figueroa WS, Drenick EJ. Tix effed after, daresof leucinc. isoleucineor valine on ularma amino acid levels.Am J ClinNutr 1965; 17 317-21. 32 SchauderP, SchrederK, Herhertz L. Lanpr K, LungenbeckU. Evidence fur valbte intokrance in patients with cirrhosis. Hep atology 1984;4: 667-70. 33 Adibi SA. Roles of branched-chainamino acids in metaboiic reeulation. J Lab Clin 1980;95: 475-84. 34 Milner RDG. The stimulation of insulin release by errential aminoacids from mbbit pancreasin vitro. J Endocrbwl 1970: 477: 437. 35 Bnchar t&R. Swaffield MN. Synorgirticaction of glucaguu and insulin in regulation of hepatic regeneration. Adv Enzyme Regul 1975; 13:28-93. 36 Takatsuki K, lwiiwara K, Hayashi S. et rd. Acceleration of DNA synthesisin postlhepatectomi&d regeneratingliver of normal rat byinsubn and glucagon.Lift Sci 1981; 29: zM)9-15.

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Effect of glucose and branched chain amino acid (BCAA) infusion on onset of liver regeneration and plasma amino acid pattern in partially hepatectomized rats.

Immediately after 70% hepatectomy, rats were infused via the jugular vein with one of the following solutions: saline, 3% or 8% glucose solution, 3% B...
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