Jejunal and ileal absorption of L-leucine has been studied in rats subjected to jejunoileal
bypass and in sham-operated rats, for five minute periods, using a perfusion technique. Aminoacid concentrations were: 1, 2.5, 5, 10 and 25 mM. In sorne experiments methionine was added to determine simple diffusion. The ratio of the active/diffusive components of absorption were calculated at the different luminal aminoacid concentrations in both groups of rats, showing that this ratio was lower in control animal

Bolufer González, José

Carreras Sánchez, Olimpia

Delgado Guerrero, María Josefa

Murillo Taravillo, María Luisa

idUS. Depósito de Investigación Universidad de Sevilla

REVISTA ESPAÑOLA DE FISIOLOGIA , 44 (3), 295-302, 1988       Leucine Absorption  after Jejunoileal  Bypass in Rats  O. Carreras*, M. L. Murillo, M. J. Delgado and J. Bolufer  Departamento  de Fisiología Animal Facu ltad de Farmacia 41012 Sevilla (Spain) (Reccived on Fcbru ary 26, 1988)  O. CARRERAS,  M. L. MUR ILLO, M. J. DELGADO and J. BOLUFER.  Leucine  Ab- sorption after Jejunoileal Bypass in Rats. R ev. esp. Fisiol. 44 (3), 295-302,  1988. J ejunal and ileal absorption of L-leucine has been studied in rats subjected to jejuno ileal bypass and in sham-operated rats, for five minute pcriods, using a perfusion techn ique. Ami- noacid concentrations were: 1, 2.5, 5, 10 and 25 mM. In sorne experiments methio ninc was added to determine simple diffusion . The ratio of the active/diffus ive components of absorp- tion were calculated at the different luminal aminoacid concentrations in both groups of rats, showing that this ratio was lower in control an imals.  Key words: Rat , Leucine, Intestina l absorption, Intestinal bypass, Leucine absorption.    The exclusion of segments of proximal and distal small bowel from normal con - tinuity by surgical bypass provides the opportunity not only to stud y th e effects of deprivation of luminal nutrition from the bypassed intestine, but also to examine the changes in the intestine that remains in continuity (9, 10). These changes are hy- poplasia (in the bypassed segment), and hyperpl asia (in the continuing intestine). In the atrophic mucosa the non-electro- lyte transpon in vivo is d iminished when expressed as absorption per cm of intes- tinal length (11), whilst in hyperplastic mucosa an increase in non-electrolytes tran spon in vivo is found when the same referen ce system is used (5, 13). On the other hand,  experiments  with isolated in-  * To whom all correspondence should be ad- dressed. testina! epithelial celis (21) or brush-bor- der membrane ves icles (12) revealed that nutrient transpon remains unaltered, in- dicating that the observed changes in vivo could be explained by alterations of the mucosa! surf ace more than by alterations of  the individual enterocytes. The aim of the  present work was  to study, under in vivo conditions, the ab- sorption of L-leucin e in rat small intestine 3 months after jejunoileal by pass in both hyperpla stic and hypoplastic intestinal segments , distingui shing between mediat- ed and non-mediated aminoacid trans- pon.   Materials  and Methods  Bypass operation.- Male Wistar rats, 3 months old  and weighing about 300 g,            1.54 ± 0.07  0.005 0.131 ± 0.008 0.001 2.9 ± 0.2 0.001  1S. 7.9 ± 0.5 0.578 ± 0.035 i     296 O. CA RRERAS, M. L. MURILLO, M. J. DE LG A DO AND J . BOLUFER LEUCINE ABSORI'TION  ANO BYPASS IN RA TS  297 maintained  on a standard pellet  diet with free  access  to  tap  water,  were  used  for these experiments.  Anim als, after a 24 h fast were anaesthetized  with  sodium pen- tobarbitone  (4 mg/ 100 g b.w.,  i.p.).  Lap- arotomy  was then  carried  out and  45 cm jejunoi leal bypass of the small bowel was the  K'0   (apparen t  mass-  transfer-coeff i- cient in nmol/cm 2/min/mmolll ), was de- termined   by   linear   regression   analysis. The   active   transport    kinetic   constants were  calculat ed  from  the curve  obtained by  fitting  the  data  of  non -passive  trans- pon with an unweighed single rectangular  Table l.  Effect of j ejunoileal bypass on intestinal structura/ characteristics of the rat j ej unum and ileum Data are means ± S.E.M. P: In comparison with the respective sham-operated . Number of rats = n.s. = not significan!.  Parameter Sham-operated Bypassed p Body weight {g) At start 358 ± 10 356 ± 11 performed  starting 2 cm from the Treitz ligament, as previously  descr ibed  by NE- METH et al (1 S). Sham operations were hyperbola (difference curve) by a devel- oped program in an Apple II Europlus computer. 1 month after operation 3 months alter operat ion  Jejunum 418 ± 6 392 ± 14 463 ± 6 413 ± 12 n.s. n.s. 0.001 performed  on  an equal  number  of  rats, whose  intesti ne was  cut  and  re-anasto- mosed without bypass. In each in- stance, continuity of the gut was restored by end-to-end anastomosis (fig. 1). After 3 months,  both  groups of animals were  Tissue morphometric evaluation.- The outer circumfere nce of the intestine was measured in situ during perfu sion as has been described by WINNE (23). The villus surface and the mucosa! surface area were Outer circumference (cm) 2.10 ± 0.05 Villus surface (mmz) Mucosa! surface (mm2/ mm2 serosa)  /l eum Outer circumference (cm) 2.25 ± 0.08 2.6 1 ± 0.08 0.001 Villus surface (mm2) 0.231 ± 0.011 used for absorption experiments. calculated  using the equation developed Mucosa! surface (mm2/ mm2 serosa) 5.9 ± 0.3 0.437 ± 0.022 0.001 In vivo absorption.- Rats we re starved for 24 h and anaesthetized  with sodium by ECKNAUER et al (8). Statistics.- The results are presented as   6.6 ± 0.3  n.s.   pentobarbitone (4 mg/ 100 g bod y wt, s.c.). Inflow and outflow cannulae were tied into jejunum and ileum; thus, jejunal and ileal loops about 20 cm were isolated from continuity with the lumen. The loops were rinsed with O.9 % NaCI so- lution, replaced inside the body wall and perf used ata flow rate of 5.6 mllmin with prewarmed saline containing (mM): Na+, 150; CJ-,  135; HCO.J,  15; K+ , S  and H2P04, 5 (pH = 7.4). An imals were maintained under controlled temperature. Multiple-pass perfusions of jejunal and ilealloops with saline solutions containing H3-labelled L-leucine or both L-leucine and methio nin e, over a range of concen- trations, during a period of 5 mio, were carried out. The absorption of the ami- noacids was measured as luminal loss and the average of  the ind ividual mean s with the standard error of the mean indicated. The significance of diff erences was deter- mined by stand ard paired t test.  Materials.-   L-(4,5-3H)   Leucine   was f rom Amersham Intern ation al, and L-leu- cine and methionine we re from Sigma. These and al! other reagents were of A.R. grade.   Results  The body weight of sham-ope rated rats was significandy higher than that of by- passed animals, at three month s from the surgica l operation (Table I).  Intestinal tissue.- After 3 months of 45 area  si n_ificantly  dec eased _in  the  by- passed  ¡e¡unum  and  shghtly  mcreased  in the ileum.  Leucine _    absorption_    by   sham   and   by- passed  ammals.- JeJunal  aminoacid  ab- sorption   by   sham   and   bypassed   rats measured  in  the presence  and  absence of L-methionine at, 1, 2.5, S, 10 and 25 mM concentrations in the bulk phase and re- lated to s_erosa! surface is shown in fig. 2. The rela wnsh1p betwe n total absorption and leu cme concentratJon was non-linear in sham and bypassed jejunum  (Curves A,      A -ILEU M SHAM -- CONT JNUING ILEUM       'E .\¿ 20C was expressed  in nmoles/cm2  serosa! sur- face/min. cm jej un oileal by pass, the continuing ileum was hyperplasied and the bypassed jejunum was hypoplasied (table I). Valu es ---•Ce' Kinetic analysis.- The character ization of the intestinal absorption process rests upon the determination of the contribu- tion of passive and non-passive compo- nents.  The  passive  component  was  ob- tained  in the presence of methionine,  and    Rev. esp. Fisiol., 44 (3),1988 for outer circumference and villus surf ace area showed statistical differences between sham and bypassed animals, in both je- junum and ileum. These values decreased in the bypassed jejunum and increased in the continu ing ileum. The mucosa! surface     Fig. 1. lnt stirz_al bypass used in this study . End -to-end  wtth  deocolostom y.  Hatching  zone: bypassed  segment.   Rev. esp. Fisiol., 44 (3), 1988 25- lEUCINE CONCENTRA TrON. mM.  Fig.  2. ]ejunal  leucine absorption  by sham-oper- ated and bypassed  rats (dotted line). Cu rve A and A', total absorption; cu rve B and 8', passive comp<?ncnt ; cu rve C and C' non -pas- stve com poncn t.  Sham 25.9 ± 0.5 5.3 ± 0.1 146 ± 3 3.3 ± 0.1 5.2 ± 0.2 18 ± 0.5 Bypassed 19.3±1.1* 14.1 ± 0.6* 234 ± 5' 6.6 ± 0.4* 13.9 ± 0.6 80 ± 6* /le um Sham  21.6 ± 0.7  6.3 ± 0.2  210 ± 15  3.8 ± 0.1  6.3 ± 0.2  35 ± 4  S .. . B :o, - > ¡:     298 O. CARR ERAS, M . L. M URI LLO, M . J . DELGADO AND J. BOLU FER  Table  11.     Effect of jejuno ileal bypass on kinetic parameters  of /euc ine absorption referred lo serosa/  and mucosa/ surface. Data represents means ± S.E.M. tor fiheen animals. • P < 0.001. K'o = nmol/cm2 · min ·mM. K'M = mM.    1.6  1.4 LEUCINE  A BSORPTION  ANO  BYPASS  IN  RATS  299  Discussion A JEJUNUM  SHAM - vation of luminal nutrition, either by star-      Jejunum     K'o  J 'max = nmol/cm2 · m1n.  Serosal Mucosa!  K'.., J'max K'o K'" J 'maJC o A' ¡: "w' ;>;; ¡¡ ou. ;;¡. ...._ ...,    JEJUNU M  BYPASSED  ILEUM  SHAM  CONTINUINC ILEUM  There is growing evidence that depri-  vation or by surgical exclusion, leads to progressive structural atrophy  (7,  17). Our f indings are in accordance with the (J   04 ....... .8 ' results  report ed  by different authors after bypass  (2,  14). The  outer  circumference    Cont inuing 17.9 + 0.5*   10.3 + 0.3* 248 + 24 2.7 + 0. 1 *    10.2 ± 0.3* 38 ± 5   0.2 • 12.5 S 10  25 LE UC INE  CONCENTRAT ION    mM. (cm),  villus  surface  (mm 2)   and  mucosa! surface  (mm 2/mm2   serosa)  decreased   in bypassed je j unum as compared with sham je j unum (table I). In the continuing ileum , opposite struc-   A'). However, in the p esen e of 60 mM methionine a linear relauonsh1p was found in both an imal groups (Cu ves B, B') re- vealing that this concentr uon as an ef- fective inhi bitor of L-leucme act1ve trans- port. Th e slope of this !in gives the which is the apparent pass1ve. permeabdJt y coeff icient for the non-med tated compo- nent. The results reveal that K'o was smaller in by passed animals (table ll). Mediated  leucine  transport  was  calcu- lated by the diff erenc between total ab - sorption and non -m ed1ated transp?rt .(ab- sorption in the presence of methwm':l e). When these va lues were plotted agamst the concentration  of leucine in the ?ulk phase   saturation  curves  were  obtamed (Cur;es C, C'). In the sham jejunum  the saturable component represents 46 %. of the tota l absorption  at 1 mM  L-leucme and only 16 % at 25 m!Vl L-l ucine. This component increased shghtly m bypassed j eju nu m  (50 %  and  23 %  respec uvely) . The apparent kinetics constants (K'M a!1d J'max), calculated by fittin g the data wJth   tained (table II) : the K'I? in byps ed je- junum was higher than JO sham ¡e¡u num and J'max became abo 5-fold .high er in bypassed than in sham ¡e¡unu m, mstead of 1.7-fold found when the results were ex- pressed as serosa! surface: Ileal aminoacid absorptwn by sha.m and bypassed  animals was r:neasured . m. he same  experimental  condmons  as m  ¡e¡u-    -J EJUNUM SHA M - JEJUN UM BYPASSED Fig. 4. Active!Diffusi ve ratio of L-leucine j ejunal (A) and ileal (B) absorption in sham-operated and bypassed rats.   num . Results show a decrease in total ileal leucine absorpt ion after jejunoileal bypass when referred to serosa! or mucosa! sur- face (fig. 3), but an increase wh en referred to intestinal length (data not shown). The passive component was a linear function of the su bstrate concentration, whose slopes have greater values in sham oper- ated animals than those obtained in by - passed animals (table II). In  the  range  of  leucin e concentrations studied in the sham ileum, the non-passive component accounts for 56 % of the total absorption  at   1  mM  leucin e,  and  only 23 % at 25 mM leucine concentration (fig. 3).  This  component  was  similar  in  the continuing ileum of the bypassed  animals (52 %  and  28 %  respecti vely). After  je - junoileal   bypass,   the  continuing   ileum showed  an  increase  in  K'M  and  J 'max, similar levels remaining in sham and by- passed  animals  both  expressed  as serosa! tural changes were found , showing that mucosa were hyperplastic, in accordance with others (6, 12, 16). The absorption in bypa ssed jejunum 3 months after bypass, when both mor- phological and functional changes had reached the final stage, was studied. We found a decrease with respect to sham tis- sues in the K'0 valu es that are higher than those reported in vitro studies (4) and a significant increase in the kinetic param - eters, verif y ing that the control K'M values were qu i te similar to those determ ined in vivo (1, 3, 18, 19). In view of the fact that until  now  the individual  enteroc y te has been   found   to  be   unchanged   in   the atrophic  mucosa  (14, 20),  our  findings must be interpreted as indicative of an in - creased number of transpon sites in the individual enterocytes or of a larger pro- portian of epithelia l cells in the mucosa of bypassed loops (14). Since, after jejunoileal bypass, a slight increase in mucosa! surface has been shown (table 1), the results of ileal leucine absorption, when referred to mucosa! sur- face, revealed that K'0 decreased and K'M an unweighed  single rectan g lar  hyper-    surf ace area  and  as  mucosa! surface area increased after bypass . Regarding the dif- bola,  significantly   inc e sed .JO    the  by- passed    j eju num    after   ¡e¡unodeal    bypass 1 2.5    S  25 L E UC!NE  CCNCENTRATION . mM. (table II). The ratio of active (methionine-sensi - ferences in K'0, terpreted  by these results should be in- into account that in (table II). . When  these pr evwus  results were  ex- p ressed with reference to muco.sal surface, taking  into  account  the data  m  Table  1, new va lues of  K'0  and J 'max  were ob-   Rev. esp. Fisiol., 44 (3). 1988 Fig. 3. Ilealleucine  absorption  by sham-operated and bypassed rats (dotted fin e) . Curve A and A', total absorption ; cu rve B and B', passive component ; cu rve C and C' n on-pas- sive component. tive)/diffusive       (meth ionine-insensi tive) absorption indicares i n both jejunum or ileum of sha m  or bypassed rats, that the active pathway is more important at low luminal am inoacid concentrations (fig. 4).   Rev. esp. Fisiol., 44 (3),1988 taking the atrophi c (bypassed jej unum) or hy- pertrophic (continuing  ileum ) mucosa, changes in the un sti rred water layer thick- ness can be expected. In accordance with the equation developed by WESTERGAARO  300 O. CARRERAS, M . L. MURILLO, M. J. DELG ADO AND j. BOLUFER LEUC INE ABSORPTION  AND BYPASS IN RATS  301 and DIETSCHY  (22), the higher the un- stirred water !ayer thickness, the greater the bias in the determination of the values of K'0, so that the real differences in this parameter should be more attenuated than those shown for jejunum and ileum in Ta- ble III. The same reasoning can be applied to values of K'M and J'max. In this case a higher unstirred water layer thickness should produce a higher decrease in the K'M and a higher increase in the J'max val- ues, in accord ance with the theoretical model developed by WINNE (24).  Thus, the real differences regarding K'M values should be more attenuated in the ileum and more prominent in the jejunum, whereas J'max values should be the op- posite. It i s known that in the interdigestive pe- riods, non-electrolites can leak into the lu- men and its reuptake occurs by active transport. The bypassed jejunum, with its normal blood flow, can be considered to be permanently in the interdigestive pe- riod, so an increased affinity and capacity in the aminoacid active transpon system could be more efficient. On the other hand, the ratio active/d if- fusive components slightly chan ged in je- junum and ileum of bypassed animals, re- vealing that for the leucine concentrations used, this ratio was lower in control ani- mals.  Ackno wledgment  This study was supported by a Grant PBSS-0331 from the Comisión Asesora de Investigación Cien- tífica y Técnica, Spain.   Resumen  Se estudia la absorción intestinal de L-leucina (1, 2,5, 5, 10 y 25 mM ) en yeyuno e íleon de ratas so- metidas a un bypass yeyunoileal y en patrone s (ratas sham), utilizando técnicas de perfusión durante 5 min. Se utiliza en algunos experimentos la metionina, para determinar la difusión simple. La relación de los componentes activo/difusivo de la absorción se cal-   Rev. esp. 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Leucine absorption after jejunoileal bypass in rats

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Leucine absorption after jejunoileal bypass in rats

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idUS. Depósito de Investigación Universidad de Sevilla