Gut 1997; 40: 67-72

Enteral supplementation with ornithineo ketoglutarate improves the early adaptiveresponse to resection

B Czernichow, E Nsi-Emvo, M Galluser, F Gosse, F Raul

AbstractBackground-Polyamine synthesis oruptake, or both, might be an importantevent that initiates the adaptive hyper-plasia seen in the intestinal remnant afterpartial small bowel resection.Aim-The ability ofan enteral diet supple-mented with the ornithine salt: ornithinea ketoglutarate (OKG), a precursor forpolyamine synthesis, to modulate theadaptive response of the remnant ileumafter jejunectomy was evaluated.Methods-Adult Wistar rats underwent aresection ofthe proximal 50% of the smallintestine. Controls underwent a singletransection. The rats were fed intra-gastrically with a nutritive mixture sup-plemented either with casein hydrolysateor with OKG (1 g/kg). The isoenergeticand isonitrogeneous diets was given con-tinuously for seven days.Results-Villus and crypt hyperplasia wasobserved in the remnant ileum comparedwith transected controls. OKG supple-mentation started after resection a furtherincrease in villus height. After resection,OKG supplementation increased signifi-cantly the putrescine content and theamount of ornithine decarboxylasemRNA. A twofold to threefold increase ofsucrase activity was measured in the re-sected animals compared with thetransected rats. In contrast, the amount ofsucrase mRNA was significantly lower inthe ileum of the resected rats and OKGsupplementation initiated a further dropin the amount of sucrase mRNA withoutpronounced changes in enzyme activity.Conclusions-The adaptive hypertrophyseen after resection can be accelerated bysupplementing the diet with ornithine(OKG) a precursor of polyamine syn-thesis. In the remnant ileum, the reducedamount of sucrase mRNA, despite theincreased level of sucrase activity,suggests a post-translational control ofsucrase expression.(Gut 1997; 40: 67-72)

Keywords: jejunectomy, intestinal adaptation,putrescine, sucrase, ornithine decarboxylase,intragastric feeding.

After partial small bowel resection, theremnant intestine undergoes compensatorystructural and functional changes characterisedby villi hyperplasia, increased crypt cell pro-

liferation, and increased migration rate ofenterocytes along the villi, which combine toproduce improved digestive capacities."Many studies have shown that enteral feedingcan stimulate the adaptive response of theremaining intestine and that the extent ofintestinal response depends on the content ofthe diet.9 Although the events governing theadaptive response to resection are poorlyunderstood, numerous factors are known tostimulate epithelial cell proliferation anddifferentiation and may potentially have atherapeutic role in the treatment of short bowelsyndrome.8 1012 In this regard, several studieshave suggested that polyamine synthesis oruptake, or both, might be an important intra-cellular event, which initiates the adaptivehyperplasia seen in the intestinal remnant afterpartial small bowel resection.'3-1' Indeed, thepolyamines (putrescine, spermidine, spermine)modulate RNA polymerase activity and stimu-late RNA, DNA, protein, synthesis, celldivision, and tissue growth.'5 The key of poly-amines seems to be corroborated by thefindings that the inhibition of mucosal orni-thine decarboxylase (ODC) activity with thespecific inhibitor difluoromethyl ornithine(DFMO) prevents intestinal adaptation aftersmall bowel resection in rats.'6 Both enteraland intravenous putrescine and spermidinecompletely restore adaptive mucosal growth.'6In addition, it has been reported that theinhibition of diamine oxidase, the polyaminedegrading enzyme, by aminoguanidinetreatment improved the adaptive response tointestinal resection.'7 18As an alternative, the stimulation of the

endogenous synthesis of polyamines in themucosa of the remnant intestine would repre-sent another approach that may potentiatepost-resectional gut adaptation. In this regard,a dietary supplementation with the amino acidornithine, the direct precursor of putrescine,might represent an interesting candidate toimprove the post-resectional adaptive process.The highly soluble ornithine salt: ornithine cxketoglutarate (OKG), is formed of two mole-cules of ornithine and one molecule of ot keto-glutarate. It efficiently improves the nutritionalstatus in protein depleted patients,'9 and hasbeen successfully used enternally and intra-veously in the treatmnent of surgical patients20 aswell as in burned and traumatised patients.2122In healthy subjects, oral administration ofOKGproduces a transient increase in plasmaconcentrations of ornithine and a rapid increasein urea excretion, which suggests that thecompound is metabolised extensively.23 The

CJF INSERM 95-09,IRCAD, Strasbourg,FranceB CzemichowE Nsi-EmvoM GalluserF GosseF RaulCorrespondence to:Dr F Raul,CJF INSERM 95-09,IRCAD, 1, place de l'h6pital,BP 426, 67091 Strasbourgcedex, France.

Accepted for publication28 August 1996

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small intestine is a major site of metabolism forornithine.24 We have previously reported that anenteral supplementation with OKG given tohealthy rats stimulates the decarboxylation ofornithine by ODC leading to an increasedsynthesis of putrescine in the intestinalmucosa." The aim of this study was thereforeto evaluate, in the rat, the ability ofOKG givenenterally to modulate adaptation of theremaining ileum after 50% resection of thesmall intestine.

Methods

AnimalsThe experiments were conducted according tothe National Research Council Guide for useand care of laboratory animals with the author-isation (n°00573) of the French Ministry ofAgriculture.Male Wistar rats (n=28), weighing 400-430

g, were housed under standardised conditions(22°C, 60% relative humidity, 12 hours lightand 12 hours dark cycle) and were randomlydivided into two groups. The animals from theexperimental group (n= 14) underwent partialresection of the small intestine as follows. Therats were anaesthetised with an intraperitonealinjection of Ketamine (Sigma, UK) (0-5 mg/100 g body weight), the abdomen was openedthrough a midline incision. The length of theintestine was measured from the ligament ofTreitz to the ileocaecal junction. The proximal50% of the intestine was resected. The duo-denum was anastomosed to the remainingileum in continuity. Control rats (n= 14)underwent anaesthesia, laparotomy, and asingle transection at the middle of thejejunoileum followed by reanastomosis.

After surgery, the animals were starved for24 hours and thereafter fed enterally via anintragastric catheter.25 They received a nutri-tive mixture (Normoreal Na4O, LaboratoriesSodietal, France) containing (in terms ofenergy): 15% protein (50% casein and 50%soya protein), 55% carbohydrate (malto-dextrin), and 30% soya oil. The enteral mix-ture was administered continuously at a flowrate of 3-4 mi/hour and provided a daily calorieintake of 82 kcal per rat. All animals had freeaccess to drinking water.The resected animals and the transected

controls were given continuously for sevendays the enteral mixture supplemented eitherwith free aminoacids (casein acid hydrolysate,Sigma, UK, 1 g/kg per day, n=7) or with theornithine salt (OKG, laboratories J Logeais,France, 1 g/kg per day, n=7).25 The diets wereisoenergetic and isonitrogenous, and containedthe same amounts of electrolytes and vitamins.One hour after the end of the enteral feeding

period (seven days), a 20 cm segment corres-ponding to the terminal part of the ileum wascollected under ether anaesthesia.

HistologyIntestinal samples corresponding to the first 2cm of the collected intestinal segment were

fixed in 10% formalin. The fixed samples weredehydrated in progressive concentrations ofethanol, cleared in xylene, and embedded inparaffin wax. Deparaffinised 5 ,um sectionswere stained with haematoxylin and eosin. Thevillus length and crypt depth were measuredwith an image analyser. Only villi sectionedfrom their base to the top with a singleepithelial layer at their tips and crypts, and avisible lumen along their entire depth wereconsidered as suitable for quantitativeevaluation.

BiochemistryImmediately after collection the intestinalsegment was flushed with ice cold 0 9% NaCl.The mucosa was scraped off with a glass slide,weighed, and frozen in liquid nitrogen.Enzyme assays - samples of the mucosa were

homogenised in mannitol (50 mM), TRIS 2mM (pH 7*1). Brush border membranes wereisolated from mucosal homogenates asdescribed by Schmitz et al.26 Sucrase activitywas determined according to the method ofDahlqvist.27 Enzyme activities were expressedeither as specific activities in the purified brushborder membranes (mU per mg of protein) oras total activities per segment (mU per cm ofintestinal length). One unit of activity corres-ponds to one p,mol of product formed perminute at 37°C.

Polyamines - aliquots of the mucosal sampleswere homogenised in 10 parts (w/v) of per-chloric acid (200 mM), and the homogenateswere centrifuged at 3000 g for 10 minutes afterstanding for 16 hours at +2°C. The clearsupernatants were diluted with perchloric acid(200 mM) and 200 ,ul aliquots were applied ona reversed phase column for separation. Thepolyamines were determined by separation ofthe ion pairs formed with n-octanesulphonicacid, reaction of the column effluent witho-phtaladehyde/2-mercaptoethanol reagent,and monitoring of fluorescence intensity.28RNA isolation and northern analysis - total

cytoplasmic RNA from the ileal segment wasextracted by acid guanidinium thiocyanate-phenol-chloroform extraction.29 For northernblot analysis, aliquots (20 ,ug) of total RNAwere separated by electrophoresis on 1%agarose, 17% formaldehyde gels, andtransferred to nitrocellulose filters. The filterswere hybridised to 32P-labelled nucleotideprobes for 1 actin30 used as internal standard,sucrase-isomaltase3l and ODC32 according tostandard procedures.33 The filters wereexposed to Kodak XAR-5 films using anintensifying screen. Specific mRNA wasquantified by densitometric scanning of thebands. Correction for loading was performedby dividing the densitometries of these bandsby the densitometries of the interal standard (,Bactin) used as reference.

StatisticsData are reported as means (SEM). Statisticaldifferences between groups were evaluated byone way analysis of variance and specific

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Enternal nutrition with ORK and response to resection

Body weight changes and total mucosal wet weight of theintestinal segment (20 cm length)

Body weight MusocalDiet group changes (g) weight (g)

Transected ratsCasein (7) -11 (5) 0-77 (0-05)*OKG (7) -17 (2) 0-80 (0-06)*Resected ratsCasein (7) -17 (2) 1-34 (0 06)tOKG (7) -20 (4) 1-33 (0 07)t

Transected and resected rats were fed an enteral dietsupplemented with casein hydrolysate (1 g/kg per day) or withornithine Cs ketoglutarate (OKG) (1 g/kg per day). Values aremean (SEM). The number of animals per group is inparentheses. *Compared witht, p<0-01.

differences were identified using Student-Newman-Keuls multiple comparisons test.

ResultsAs the Table shows, the body weight changeswere not significantly different between thevarious groups. After seven days of enteralfeeding the body weight loss of the transectedand resected animals was very similar and didnot exceed 5% of their initial weight. Themucosal weight of the ileum was increased by65% in the resected animals when comparedwith the transected controls, the modificationsin the composition of the enteral diet had noinfluence on those changes.

In the resected animals, after seven days ofenteral feeding, villus hyperplasia andincreased crypt depth was observed in theremnant ileum compared with the transectedcontrols (Fig 1). In the resected animals, OKGsupplementation caused a further increase inthe height of the ileal villi without significantchanges in crypt morphology compared withthe resected rats receiving casein hydrolysate.The putrescine, spermidine, and spermine

content (Fig 2A) and the amount of ODCmRNA (Fig 2B) were significantly increased in

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Figure 1: Villus height and crypt depth in transected andresected animals. Transected rats werefed enterally a dietsupplemented with either casein hydrolysate (open column),or with ornithine a ketoglutarate salt (dotted column).Resected rats werefed enterally a diet supplemented witheither casein hydrolysate (black column), or a dietsupplemented with ornithine a ketoglutarate salt (hatchedcolumn). Values are means (SEM), n=7per group. Forvilus or crypt, columns not sharing a common superscriptdiffer significantly; p<0 05.

the ileal mucosa after jejunectomy. OKG sup-plementation increased significantly the putres-cine content and the amount of ODC mRNAin the mucosa of both transected controls andresected animals when compared with thecorresponding rats receiving casein hydrolysate.OKG supplementation caused about a twofoldincrease in the amount of putrescine and in theexpression ofODC mRNA in the ileal segmentafter jejunectomy when compared withresected animals receiving casein hydrolysate.However, under those conditions, the amountof the other polyamines: spermidine andspermine, were not increased.The total activity of sucrase, expressed per

cm intestinal length, was significantly in-creased in the remaining ileum after jejunec-tomy after seven days of enteral feeding. Simi-larly, a twofold increase of the enzyme specificactivity in the brush border membranes wasalso observed (Fig 3A). OKG supplementationdid not modify the level of the total enzymeactivity measured in the remaining intestinalsegment after resection. At the cellular level,OKG supplementation caused a significantdecrease in the level of specific activitymeasured in the brush border membrane of theenterocytes lining up the villi. Changes in theamount of sucrase mRNA (Fig 3B) did notparallel those seen at the level of enzymeactivity. In contrast with the increased enzymeactivity, the amount of the correspondingmRNA was significantly lower in the ileum ofthe resected animal compared with thetransected controls. OKG supplementation ledto a further significant decrease in the amountof the mRNA in the ileum. This effect wasprominent in the mucosa of the remnant ileumof the resected animal despite the maintenanceof high level of enzyme activity.

DiscussionIncreased ODC expression and polyaminecontent play a crucial part in the adaptiveresponse of the remnant intestine afterresection.1316 This study shows that feedingrats with an enteral diet supplemented withOKG, a precursor of endogenous polyaminesynthesis,'9 25 increases ODC expression, in-creases the putrescine content of the mucosa,and accelerates mucosal growth in the ileumafter jejunectomy. However, the effect ofOKGon polyamines was restricted to putrescine asneither spermidine, nor spermine were in-creased by the precursor administration. Thisspecific effect ofOKG supplementation on theputrescine content of the mucosa has also beenobserved in healthy animals and results fromthe balance existing between the two metabolicpathways that are activated by OKG treatmentin the intestinal mucosa for polyamines: thedecarboxylation of ornithine by ODC and theoxidative deamination of putrescine to -yaminobutyric acid.25

Intestinal adaptation after resection seems todepend on a number of factors. It has beenshown that a potent nutrient independentstimulus for intestinal cell proliferation occurswithin minutes after resection and that the first

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Figure 2: (A) Polyamine content of the ileal mucosa in transected and resected animals. Values are given in nmol/g wetweight (means (SEM), n= 7 per group). For a given polyamine: columns not sharing a common superscript differsignificantly; p<005.) (B) Relative amount of ODC mRNA in the ileal mucosa of transected and resected animals. Theamount ofmRNA measured in transected control rats were used as reference. The amount ofmRNA was quantified bydensitometric scanning of the bands. Correction for loading was performed by dividing the densitometries of these bands bythe densitometries of the internal standard. Values are means (SEM), n=7 per group. Columns not sharing a commonsuperscript differ significantly; p<005. Transected rats were fed enterally a diet supplemented with either casein hydrolysate(open column), or with ornithine ot ketoglutarate salt (dotted column). Resected rats were fed enterally a diet supplementedwith either casein hydrolysate (black column), or a diet supplemented with ornithine oa ketoglutarate salt (hatched column).

targets of this stimulus are the crypt cells in theresidual intestine.4 However, other studies haveshown the importance of luminal nutrients inthe adaptive processes to resection.34 35 Inaddition, this study suggests that the compo-sition of the diet given enterally modulates thedegree of adaptation, which is time depen-dent.35 The early adaptive response toresection (that is, in the first week) seemsdependent on changes in polyamine metab-olism and leads to increased mucosal mass andhyperplasia. This effect seems at least partlyrelated to mucosal exposure to nutrients,5 6 asshown here with OKG supplementation.However, it is obvious that the complex mech-anisms involved in the adaptive responses toresection are also dependent on severalsystemic or local factors.12 36At a functional level, mucosal disac-

charidases are reported as increased, un-changed or even decreased in the remnantintestine after resection; however, most studiessuggest that the total activity of brush borderenzymes increase in parallel to the increase inmucosal mass.2 7343738 Our data show anincrease in brush border sucrase activity in theresected animals that is concomitant with theincrease in mucosal mass and hyperplasia ofvillus and crypt compartments. Resectioninduced in one week a twofold to threefoldincrease in sucrase total and specific activities

in the remaining ileum, indicating increaseddigestive capacities for the ileal segment afterresection.The level of sucrase activity in the brush

border membranes is often used as a bio-chemical marker of functional maturity of theenterocyte. However in the light of our data,the role of sucrase activity as a marker ofenterocyte maturity becomes questionable.Indeed, after resection we report an earlyincrease in both total and specific activity ofsucrase despite a twofold decrease in theamount of sucrase mRNA in the remaining ilealmucosa. Several trends of evidence support theview of increased enterocyte immaturity in theremaining ileum after resection: hypertrophiedvilli with reduced life span, increased rate ofcrypt cell proliferation,39 higher level of ODCexpression, increased polyamine synthesis, andreduced expression of sucrase transcripts. Toconciliate the increased enterocyte immaturitywith the higher level of sucrase activitymeasured in the ileum after jejunectomy, thepossiblity of a post-translational control ofsucrase expression should be considered. Thetrigger to the post-resectional increase insucrase activity is the sudden exposure of theileal mucosa to carbohydrates that werepreviously absorbed upstream. These sub-strates, resulting from intraluminal hydrolysisof starch by pancreatic enzymes, may stabilise

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Enternal nutrition with ORK and response to resection

Segmental activity

b 700

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Figure 3: (A) Sucrase total activity in the mucosa per centimetre intestinal length an

sucrase specific activity in isolated brush border membranes in the ileum of transectedresected rats. Results are means (SEM) ofseven rats per group; columns not sharingcommon superscript differ significantly; p<005.) (B) Relative amount of sucrase m.

in the ileal mucosa of transected and resected animals. The amount ofmRNA measutransected control rats was used as reference. The amount ofmRNA was quantified Idensitometric scanning of the bands. Correction for loading was performed by dividindensitometries of these bands by the densitometries of the internal standard. Values ar

means (SEM), n=7 per group. Columns not sharing a common superscript differsignificantly; p<O 05.

the enzyme molecule (sucrase-isomaltathe brush border membrane and nenzyme processing and consequently stirits activity. It must be emphasised that s

present an inherent low expression in theunder normal physiological conditions.4enzymic response of the distal small boiproximal resection would be a resLincreased luminal nutrition acting at theof the enzyme molecule anchored in theborder membrane, a process that ispendent of the maturity status of the e

cytes lining up the villi. However trophic fsuch as hormones, regulatory peptiddietary polyamines4' could also mo

structural and functional maturationIndeed in this study we show that trea

with OKG, a precursor of putrescine syn

induced a drop in the expression of s

mRNA in the transected animal and e

bated the drop of this transcript in the n

of the resected animal indicating clearlother factors, independent of the comp(of luminal nutrients could modulatexpression of sucrase.

In conclusion, early adaptational I

trophy of the ileal mucosa after jejunectchighly dependent on polyamine metatand can be accelerated by supplementirdiet with a polyamine precursor like OKC

border adaptive hypertrophy results in an increase invillus height, crypt hyperplasia together with areduced expression of sucrase mRNA asso-ciated with increased sucrase activity in thebrush border membrane of the enterocytes.This 'paradoxal' increase in sucrase activity inthe remaining ileum might be related to theexposure to substrates that are normallyhydrolysed and absorbed upstream by theproximal part of the intestine, and which arecontrolling enzyme molecule processing andstability at the level of the brush bordermembranes. To improve better functionaldifferentiation, nutrition might represent a wayto promote a number of common adaptiveresponses that could counteract the effects ofcrypt hyperplasia and villus hypertrophy in theremnant intestine after resection.

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