Oral glutamine supplementation benefits jejunum but not ileum

PE HARDY, KL MADSEN, 0G THURSTON, SM HAMILTON, N CUI, RN FEOORAK. Oral glutamine supplementation benefits jejunum but not ileum. Can J Gastroenterol 1994;8(2):108-114. Glutamine is the primary metabolic fuel of the small intestine. The ability of enteral glutamine to support jejuna! architecture and metabolism is well established, but its effect on intestinal absorptive function, especially in the terminal ileum, remains undetermined. The purpose of this study was to develop a functional ileal fluid absorption surgical injury model and to determine if oral glutamine supplementation would be beneficial in accelerating healing and restoring function. The effects of either 1 cm resection and ilea! end-to-end anastomosis or sham laparotomy on rat in vivo fluid absorption at study start (day 0), one and two days was investigated. ln sham-operated rats, fluid absorption was not altered. In contrast, ilea! fluid absorption was significantly reduced at days O ( 17 .2±4.8 μL/cm/h) and 1 (31.4±13.6 μL/cm/h), but returned to normal by day 2 (71.0±6.2 μL/cm/h) in anastomosed rats. To examine the effects of glutamine in this model, rats were fed either glutamine (2.4 g/kg/day) or an isonitrogenous glycine-supplemented elemental oral diet for five days before their randomization to sham or anastomotic groups. This dose of glutamine reached the ileum and was completely absorbed along the small intestine. Glutamine-fed rats demonstrated no difference in recovery of in vivo ilea! fluid absorption, ilea! villus morphometric measurements, mg DNA:mg protein ratio, degree of inflammation or glutaminase activity. In contrast, jejuna!, but not ilea!, villus morphometry, mg DNA:mg protein ratio and glutaminase activity were increased in glutamine-fed 'not operated' rats (P<0.01), indicating that the jejunum, but not the ileum, responded to the glutamine-supplemented diet. These studies demonstrate that ilea! resection and anastomosis causes transient impairments in in vivo fluid absorption, and oral glutamine supplementation offers a beneficial effect to jejunal, but not ilea!, intestinal mucosa. These results suggest that the effects of oral glutamine may be limited to the proximal intestine. (Pour resume, voir page 109)

G LUTAMINE IS A NONESSENTIAL amino acid, sufficient quantities of which are synthesized by the body in healthy subjects.During surgical stress or critical illness, however, the body's requirement for glutamine frequently exceeds its production (1-4).As well, glutamine is a preferred energy substrate for small intestinal enterocytes (5)(6)(7)(8).Therefore, under the stress of a systemic illness or intestinal injury, enterocytes may be unable to meet their requirements for glutamine as a primary metabolic fuel, and exogenous ad ministration of glutamine may, therefore, be necessary.
While glutamine administration has been shown to improve jejuna!morphometric measurements in several animal models of enterocolitis (9)(10)(11)(12)(13), the effects of glutamine on ilea! morphology and functional absorption have not been well studied.Parenteral glutamine administration has been demonstrated to be safe ( 14) and beneficial (15) for humans, and it is possible that the enteral route may impart additional positive effects to the intestine.It is, therefore, important to know whether the beneficial jejuna!effects of orally administered glutamine also occur in the ileum, and whether these beneficial effects occur during typical surgical anastomotic injury or are limited to occasions of experimentally induced enterocolitis or normal tissue.
Les supplements oraux de glutamine : avantageux pour le jejunum mais non pour l'ileon RESUME: La glutamine est le principal carburant metabolique du pet it intesrin.
Ces etudes demontrent que la resection ileale et l'anastomose provoquent une alteration transitoire de l'absorption liquidienne in vivo et que le supplement oral de glutamine offre un avantage pour la muqueuse intestinale jejunale mais non ileale.Ces resultats donnent a penser que Jes effets de la glutamine orale peuvent se limiter a l'intescin proximal.
T he purpose of chis study was co determine whether supplemental oral glutamine wou ld restore deal intestinal absorptive function and improve morphometric analysis in a surgical injury model (see pan l -tl eal end-to-end surgical anastomotic mode l).In add inon, we determined whether oral glutamine supplements conferred benefits, as determined by morphomecric analysis, on the jejunum and/or the ileum in a nonsurgical model (sec part II -nonsurgical model).

ANIMALS AND METHODS
Animals: Male Sprague-Dawley rats weighing between 230 and 270 g (B1otron) were used in all experiments.The animals were allowed to become acclimatized to the animal facility for at lease 48 h before enten ng the study.Animals were housed in individual cages, on a 12 h light/dark cycle, and allowed water ad libitum.Chemicals: G lutamine, glycine, alanine, beta-NAO (from yeast, molecular weight 633.4), 2,5-and 3,5-ADP, and glutamic dehydrogcnase (type II, from bovine liver in 50% glycerol) were purchased from Sigma Chemical Co (Missouri).L-14 C-glutaminc (9.5xl0 4 MBq/ mmol) was purchased from Amersham.A ll other chemicals were reagent grade and were purchased from Fisher Scientific.

PART I -ILEAL END-TO,END SURGICAL ANASTOMOTIC MODEL
Feeding and dietary su pplementation: Previously chow-fed animals were randomized co receive either a glucaminesupplementcd (treatment group receiving glutamine) or an isonirrogenous glycine-supplemented ( untreated group receiving no glutamine) elemental d iet.
The powdered diet was provided in containers that prevented spillage or soiling but provided easy access for ad libitum intake.Food containers were weighed and refilled daily co record oral intake accurately and co prevent degradation of nutrients.
Operative procedure: Rats were anesthetized with an intraperitoneal iniection of ketamine (80 mg/kg) and xylazine (8 mg/kg).Oxygen was given by mask, and a warming lamp was placed over the operative field.The abdomen was shaved and a midline incision made.A single dose of cefazolm (40 mg/kg) was admi nistered into the peritoneal cavity, and a 10 to 15 cm segment of terminal ileum was isolated.Under 2.5x magnification, rnesentcric vessels were ligated with 6-0 silk ties on both sides of a 1 cm segment of ileum, which was then resecced.An end-toend anastomosis was initiated over a macaroni Stent using interrupted sutures of 6-0 silk.On complenon of half the anastomosis, the scent was removed and the remainder of the anascomosis completed without the stent.Injection of a saline solution to d istend mildly the intestinal lumen confirmed a competent anastomosis without leak.The abdomen was then closed.All animals were allowed to recover in an incubator warmed to 37°C and were provided with supplemental oxygen.Sham-operated controls underwent an operative procedure that simulated the surgery described above, without the resection and anastomosis.Operative procedure: The nonsurgical model received dietary supplementation but did not receive anesthesia or surgical intervention before experimentation.

MEASUREMENTS
In vivo absorption loops: In vivo fluid absorption loops were used to assess intestinal function as previously described (16).Briefly, a 10 to 15 cm segment of ileum (straddling the anastomosis) was isolated by laparotomy.
Stool was gently expressed from the segment manually.The empty portion of bowel was tied at each end with a 3-0 silk tie; care was taken not to interrupt vascular supply.This empty closed loop was filled with 2 ml of warm saline injected through a 26gauge needle.The abdomen was closed and the animal allowed to recover for 1 h before sacrifice with a lethal injection of pentobarbitol.The closed loop of intestine was removed intact and the length measured.The segment was weighed with and without the residual luminal saline.Absorption of saline, expressed in µL/cm intestine/h, was calculated as an indication of bowel function.
Morphometric assessment: This measurement was done by a researcher blinded to experimental group.Using a computerized videoplan, morphometric parameters were measured and averaged from three separate fields of each section.Villus height, number of villi per centimetre and lamina propria area:perimeter index were evaluated as previously described (17).
DNA content: The DNA content of intestinal homogenates was measured as described by Hindgardner (18).DNA was expressed as mg DNA:mg protein.
Glutaminase activity: Intestinal glutaminase activity reflects enterocyte use of dietary glutamine.Immediately after sacrifice, the rat's intestinal segments were removed, luminal contents were rinsed with ice-cold saline, and the intestine was frozen in liquid nitrogen and stored at -70°C.The glutaminase assay was performed as previously described ( 19 ,20).Homogenate protein was measured using the Bradford method (21).Glutaminase activity was expressed as µmol/mg protein/h.Location of luminal glutamine: To ensure that glutamine was, indeed, reaching the ileum, the distribution of orally administered glutam ine was deter--Anastomosis ::,..

RESULTS: PART I -!LEAL END-TO-END SURGICAL ANASTOMOTIC MODEL
Food intake: Daily food intake during the five preoperative days was similar for each group.G lutamine-supplemented animals (treatment group) consumed 14.8±3.4g feed/rat/day (0.96± 0.2 g glutamine/rat/day and 0.0 g glycine/rat/day), while those in the glycine-supplemented group (untreated group) ingested 15.1±3.0 g feed/rat/day (0.0 g glutamine/rat/day and 0.98±0.19g glycine/rat/day).This amount of glutamine is similar to that demonstrated to have had a beneficial influence on previously described intestinal injury models (2-8).Animal body weight: Animal body weight gain during the preoperative interval was similar for both the glutamine-supplemented animals ( 7.5±2 .5 g/day) and the glycine-supplemented animals (9.6±1.5 g/day).Furthermore, this weight gain is similar co agematched animals fed standard rat chow during the same period (8.3±2.0 g/day).As well, these ileal morphomecric parameters did not change between sham-operated and anastomotic groups.Heal glutaminase activity: lleal glutaminase activity, as a measure of glutamine use by the enrerocyte, was not significantly altered by glutamine or glycine supplementation or by the operative procedure (Figure 3).Location of luminal glutamine: To ensure that glutamine was reaching the ileum, 14 C-labelled glutamine was administered and its progress through the intestine mapped.As shown in

RESULTS: PART II -NONSURGICAL MODEL
Because in vivo fluid absorption, morphometric measurements and glu-taminase activity did not benefit from oral glutamine supplementation in the surgical ilea I end-to-enJ anastomotic model despite glutamine being JelivereJ to the ileum, the authors investigated the possibility of a regional difference in the ability of the small intestine to respond ro glutamine.Ilea! in vivo fluid absorption: Dietary supplementation with glutamine (treatment group receiving glutamine) or glycine (untreated group receiving no glutamine) did not alter basal jejuna!( 156±8 µL/cm/h) or ileal (95±7) fluid absorption in the nonsurgical model relative to levels seen in chow-fed animals ( 149±11 µL/cm/h and I 02± l 2, respectively) .Morphometric assessment: Villus height significantly (P<0.05)increased in the jejunum of glutamine-supplemented animals (Figure 4).In contrast, the ileum demonstrated no increase in villus height with glutamme relative ro glycine supplementation.DNA:protein ratio: To confinn that the increased jejuna!villus height was due to an increased number of enterocytcs, DNA and protein levels in jeJunal and ilea! mucosa!homogenate:; were measureJ.There was a signifi cant (P<0.02)increase in the mg DNA:mg protein ratio in the jejunum of glutamine-supplemenced animals.Once agam, the ileum did not respond to glutamine supplementation (Figure 5).Glutaminase activity: As shown in Figure 6, glutaminase activity increased in the jejunum of the gluramine-supplemented group.In contrast, glutaminase activity in the ileum was not enhanced by glutamine.

DISCUSSION
A number of studies have investigated the effects ()f parenterally anJ enterally administered glutamine on both normal anJ injured intesti ne.The majority of these studies confirmed the beneficial effects on the jejunum ( 1-8, 10-13,22-24), whereas the effects of glucamine on the ileum have been less well described (9.25).Our study thus examined the effects of oral glutamine supplementation on the ileum in both a surgical ( ileal end-to-end anastomosis) and a nonsurgical moJel.Orally administered glutamine is absorbed in the proximal jejunum, where 85% 1~ transformeJ before entry into the systemic circulation (26,27).Studies done on rats have shown that the rates of glutamine use by the jejunum are simi-I 12 CAN J GASTROENTEROL VOL 8 No 2 MARCIi/APRiL 1994 lar whether glucamine is derived from arterial blood or gut lumen (5).Nevertheless, the importance of an orally ad-ministereJ source of glutamine is suggested by the fact that rats deprived of it develop intestin al hypoplasia (28,29).
Intestinal absorptive function around the ilea!end-to-end surgical anastomosis was impaired significantly on days O and 1 postanastomosi~ as assessed by in vivo fluid absorption.While it is not surprising that intestinal fluid and electrolyte absorptive function shoulJ be diminished at the site of intestinal anastomosis, we are unaware of this decrease having hecn previously described.While the mechanism leading to this fluid absorptive dysfunction has not been examined, it likely relates to local ischcmia and subsequent free radical or cytokine release.We have previously shown that alteration in in vivo intestinal fluid absorption is a sensitive measure of mucosa!injury.Indeed, intestinal transport abnormalities will persist for days after gross macroscopic and morphometric appearance have returned to normal (16).
Our experiment showed that oral glutaminc supplementation did not accelerate healing of the fluid absorptive impairment around an ilea[ end-to-end anastomosis.Furthermore, in this surgical model, glutamine supplementation did not enhance ileal vi llus morphometric measurements or the level of ilea! glutaminase activity; this raises the possibility that adequate amounts of glutamine may not have been presented to the ileum in this model.The total quantity of glutamine (0.96±0.2 g/rat/day) consumed was likely adequate as it was similar to the amount shown to be beneficial in other studies (3,9-l3, 30).Furthermore, our studies using 14 C-labelled glutamine as a marker confirmed that glutamine was present in the ilea!lumen (Table l).
Although glutamine supplementation has been shown to enhance ileal morphometric measurements, this enhancement occurred in conjunction with a 60% small bowel resection (31) or in normal intestine from rats supplemented with parenteral glutamine   (25).These results are unlikely to be a consequence of increased glutamine delivery to the ileum because our study demonstrated no evidence of enhanced ilcal morphomctric measurements even though equal amounts of glutamine were delivered to the jejunum and ileum following oral administration.It remains to be determined whether a beneficial effect of glutamine on the ileum requires an increased duration of glutamine contact with the intestine or the presence of specific binary or pancreatic products.lncontinuity jejunum in dogs has higher glutaminasc activity and glutaminc transport levels than those seen in an excluded Jejuna[ limb in the same animals, a difference likely due to the presence of luminal constituents (32).These observations, and our own negative ilea! results, prompted us to study the effects of oral glutaminc supplementation in a nonsurgical model to determine whether the ileum is capable of responding to nral glutamine.Thi5 part of the study examined the effects of glutamine, compared with glycine (contml), in hoth ileum and jejunum.Our results were similar to those of Sal, lourn et al (24 ), and demonstrated that glutaminc supplementation enhanced jejuna!morphometric measurements, DNA content and glutaminase activity.In contrast, glutamine had no positive effect on paired ileal measurements in the same animals.TI,ese negative results occurred despite radiolabclled demonstrations that intraluminal glutamine reached the ileum in amounts similar to that seen in jejunum (Table l ).Gluramine absorbed in the proximal small intestine is transformed in the jejuna!enterocytc, and this early transformation may alter the distal small bowel response to oral glutamine.An alternate explanation is that Lhe ileum is less sensitive than the jejunum to the effects of glutamine.ln support of this hypothesis, Tamada ct al (33) recently demonstrated that the parenteral administration of the dipeptide alanyl-gluramine enhanced villus height in the jejunum but not the ileum.The ileum may thus require 'jejunalization' hy an enteral nutrient stream to become as sensitive to glutamine as the jejunum.

CONCLUSIONS
The beneficial effects of oral glutamine supplementation may be limited to the proximal small intestine.Further studies will he necessary to clarify the mechanisms responsible for the apparent nonresponse of the ileum to oral glutamine.

FeedingFigure 1 )Figure 2 )
Figure 1) Time course of in vivo fiuid absorption at the ilea!end-co-end anascomotic site.Results are compared with age-matched, sham-operated controls at zero (2 h after anascomosis was performed), and one, two and three days postanascomosis.Ilea! anastomosis reduced in vivo fluid absorption on the day of anastomosis ( day O) and one day following anastomosis.Data are mean ± SEM (n=6).*P<0.05relative to sham-operated controls Heal in vivo fluid absorption: A time course of fluid absorption at the ilea!anastomotic site is shown in Figure l.ln vivo fluid absorption at the anastomosis was significantly decreased on day 0 (2 h after the anastomosis was performed) and day 1, relative to sham-operated controls, P<0.05.By day 2, however, fluid absorption ar the ilea!anastomotic site had returned to normal.In vivo fluid absorption at the ileal endto-end anastomotic site one day following anastomosis was not altered by oral glutamine supplementation in either anastomotic or sham-operated animals (Figure 2).Heal morphometric assessment: In the glutamine-and glycine-supplemented CAN J GASTROENTEROL VOL 8 No 2 MARCH/APRIL 1994 Oral glutamine supplementation

TABLE 1
Distribution of ra?,ioactivity in tissues following oral 4 C-glutamine administration

Table 1
Villus height in jejunum and ileum of the nonsurgical model receiving oral Rlutamine (GLN) -treatment group recetvmg glutamine -err glycine (GLY) -untreated group receiving no glutamine -supplemencac1on.Villus height increased in the 1ejun11m, Ina not the ileum, of gluiaminesupplemenced animals.Data are mean± SEM (n=6).*P<0.05relative co GLY DNA:protein ratio in jejuna!and ilea! mucosa/ homogenates of the nonsurgical model receiving oral gluramine (GLN) -treatment group receiving glutamine -or glycine (GLY)uncreated group receiving no gluramine -supplementation.There is a significant increase m the DNA:/Jrotein ratio in the jejunum, hut not ileum, of glutamine-supplemented animak Data are mean± SEM (11=6).*P<0.02relative to GLY equally JistrihuteJ hetwecn Trietz' ligament and the ileal-cecal valve.
GlatamiTlilie activity in jejunum and ileum of the nonsurgical model receiving aral glutnmme (GLN) -treatment group receiving gluwmine -or glycine (GLY) -untTeated group receiving no gluwmine -supplementacion.Glutaminase activity is increased in the jejunum, bur not ileum, of the glutamine-supplemented group.Data are mean± SEM (n=4).*P<0.05relative to GLY