J.Cell.Mol.Med. Vol 6, No 3, 2002 pp. 377-382

Diverted colorectal segments are prone to develop

trophic and inflammatory changes [1]. Diversion

colitis is related to the absence of fecal stream and

nutritional elements usually present in colon lumen

[2,3]. Although reversible with the restoring of

intestinal continuity, these changes acquire special

importance in patients whose colostomy becomes

permanent for clinical reasons, as well as in the

differential diagnosis with other inflammatory

diseases [4]. Several studies have shown the ability

of glutamine to avoid and restore epithelial changes

on small bowel wall in patients maintained in total

parenteral nutrition for long periods [5–10]. This

Effects of oral supplement of L-glutamine

on diverted colon wall

F. L. Paulo *

Colorectal Surgery Division, Department of Surgery, State University of Rio de Janeiro,

Rio de Janeiro, Brazil

Received: March 15, 2002; Accepted: August 15, 2002

Abstract

Diverted colorectal segments can present trophic and inflammatory changes. These alterations are of special importance

in the patients whose colostomy becomes permanent, as well as in the differential diagnosis with other inflammatory

diseases. This study was accomplished to quantify these alterations and to determine if oral supplement of L-glutamine

would avoid them. Twenty-six adult male Wistar rats were distributed in three groups: control, colostomized and

colostomized+L-glutamine. The colostomized group received a loop colostomy. The colostomized+L-glutamine group

received a colostomy similar to the previous group and oral supplement of L-glutamine. Partial volumes of all layers of

the colonic wall were measured by image analysis stereology. The diversion caused a decrease of partial volumes of the

mucosa and the epithelium as well, and also of the height of the intestinal crypts (p<0.05). There was an increase of

partial volumes of the lamina propria, of the submucosa and of the muscularis mucosae vs controls (p<0.05). The partial

volume of the muscularis propria didn’t show significant alteration. The supplementation of L-glutamine was effective

in preventing the atrophy of mucosa and epithelium (p<0.05), also avoiding the increase of partial volumes of the

submucosa and lamina propria (p<0.05). This supplement didn’t change significantly the muscular layers. In

conclusion, colostomy causes the atrophy of the colon wall, mainly due to the atrophy of the epithelium. The supple-

mentation of L-glutamine is able to avoid these changes.

Keywords: diversion colitis • colon atrophy • glutamine • stereology

* Correspondence to: Francisco Lopes PAULO, M.D., Ph.D.Rua Ferreira Pontes, 430, bloco 1, apt 404,20541-280 - Rio de Janeiro, Brasil.E-mail: fpaulo@centroin.com.br

Introduction

study was accomplished in order to quantify the

changes in the colon wall due to diversion and to

check if oral supplement of glutamine was able to

avoid trophic changes on the colon wall in a

diverted segment.

Materials and Methods

Animals

Twenty-six adult male Wistar rats, with an average weight

of 379.37 g, were divided in three groups. There was no

weight difference between the groups (p = NS). Group I

(n=10, control) was used to determine the normal

parameters of the colon wall. Group II (n=7, CST)

received a loop colostomy 8 cm from the anal verge, and

was sacrificed after four weeks to determine the changes

on the colon wall due to diversion of fecal stream. Group

III (n=9, CST–Gln) received a colostomy like group II,

and L-glutamine oral supplement (Glutamin®, Support

Produtos Nutricionais, RJ, Brazil), 1 g / kg body weight /

day, for four weeks, and then sacrificed to verify the effect

of glutamine on the colon wall.

All the experimental protocols were approved by

Ethic Commitee and fulfilled the requirements of “Care

Use of Laboratory Animals” published by the US

National Institutes of Health (NIH Publication No. 85-

23; revised 1985).

Surgery

A 2 cm long midline incision was performed under

general anesthesia (pentobarbital, 50 mg /kg body weight

intra-peritoneal). An 8 cm long peridural catheter was

inserted in the anus and a loop colostomy done at its

distal end. The support to the stoma was done by a non-

absorbent suture (Prolene® 5-0, Ethicon, SP, Brazil)

passed through an avascular area of the mesocolon

joining the recto abdominalis muscles. The colon was

then transversally opened 2/3 of its circumference and

sutured to the skin by absorbent sutures (Dexon® 5-0,

Davis+Geck, SP, Brazil). All animals had free access to

normal pellet food (Nuvilab CR1® , PR, Brazil) and

water during the postoperative period until sacrifice. The

segments of colon used for analysis were 2 cm long and

located between 5 and 7 cm from the anal verge. Each

segment was cut in four 5 mm long rings, opened along

their longitudinal axis, flattened on moist filter paper and

fixed in Bouin solution for 12 hours. After dehydration

the material was included in paraffin for histological

section. Sections were 4 m thick and stained by the

Gomori trichromic method. Two random fields from

each colon ring, meaning a total of 8 fields for each

animal, were studied.

Stereology

Vertical sections stereology was used in this study

[11,12]. Under x250 magnification, the colon crypts

height (Ch) and partial volumes of mucosa (Vvm),

muscularis mucosae (Vvmm), submucosa (Vvs) and

muscularis propria (Vvmp) were studied. Under x500

magnification, the partial volumes of the epithelium

(Vvepi) and lamina propria (Vvlp) were studied. The

crypts length was directly measured on the images by the

Image Pro-Plus for Windows software (Media

Cybernetics, 1994, v.1.2). This software was also used to

superpose a stereological grid to the images, in order to

determine the partial volumes of the intestinal wall

layers. The stereological grid was composed of 35

cicloid arcs and 70 test points. The partial volumes (Vv)

are a ratio between the points of the grid touching the

element in the study (Pp) and the total number of points

lying over the structure where that element is contained

(Pt). Then Vv = Pp / Pt · 100 [13–17]. Statistical analysis

was done by the Mann-Whitney test.

Results

Colostomy and associated diversion colitis change

the structure of the colon wall in terms of partial

volumes of each colon layer. These changes are

diminished by glutamine supplement. The height of

crypts (Fig. 1a) was significantly diminished on

CST group as compared to control (p<0.001) and

the supplement of glutamine was able to diminish

this change (p<0.05). The thickness of mucosa

(Vvm) was diminished (Fig. 1b) in CST group when

compared to control (p<0.001) and showed an

intermediate value in CST–Gln group (p<0.05). The

muscularis mucosae partial volume (Vvmm) raised

(p<0.01) in CST group and CST–Gln group without

statistical difference between them (Fig. 1c). The

submucosa partial volume (Vvs) increased in CST

group (p<0.001) but not in CST–Gln group when

compared to control (Fig. 1d). The muscularis

propria partial volume (Vvmp) was diminished both

in CST group and CST–Gln group when compared

to control (Fig. 1e). There was no statistical difference

between CST and CST–Gln groups (p=NS). An

example of each group can be seen in Fig. 2.

378

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J.Cell.Mol.Med. Vol 6, No 3, 2002

Fig. 1 Variation of crypts height (a) and partial

volumes of mucosa (b), muscularis mucosae (c),

submucosa (d), muscularis propria (e),

epithelium (f) and lamina propria (g) between

control, CST and CST–Gln groups. Significance

indices were evaluated by Mann-Whitney test.

p=NS

Cripts height Partial volumes of submucosa

Partial volumes of muscularis propria

Partial volumes of muscularis mucosae

Partial volumes of mucosa

Partial volumes of epithelium

Partial volumes of lamina propria

Control Diversion Diversion+glutamine

Control Diversion Diversion+glutamine

Control Diversion Diversion+glutamineControl Diversion Diversion+glutamine

Control Diversion Diversion+glutamine

Control Diversion Diversion+glutamineControl Diversion Diversion+glutamine

p<0.05p<0.001

p<0.05

p<0.001

p<0.05

p<0.01

p<0.001

p<0.001 p<0.01

p<0.001

p<0.001

p<0.05

p<0.05

p<0.001

p<0.001

p<0.001

a d

e

f

g

b

c

micrometers

On a detailed study of the mucosal layer the

epithelium (Vvepi) diminished in CST group when

compared to control. Under glutamine supplement,

the Vvepi raised when compared to control and CST

group (Fig. 1f). The partial volume of lamina

propria raised in CST group when compared with

control. In CST–Gln group that volume diminished

(p<0.001) when compared to the other groups (Fig.

1g). An example of each group can be seen in Fig. 3.

Discussion

The colorectal mucosa epithelial cells use short

chain fatty acids as a primary energetic fuel,

especially acetic, propionic and butyric acids.

These are formed by bacterial degradation of

alimentary fibers contained on fecal stream [18]. In

colorectal segments downwards colostomy, the

absence of dietary fibers on the bowel lumen

380

Fig. 2 An example of the colon wall on groups:

A - control; B - colostomy; C - colostomy+glutamine.

Magnification 250 X. Gömöry staining.

Fig. 3 Effect of glutamine supplement upon colon wall

epithelium: A - control; B - colostomy; C - colo-

stomy+glutamine. Magnification 500 X. Gömöry staining.

precludes fatty acid formation and their absorption

and utilization by the epithelial cells. Glutamine is

usually a secondary fuel source for those cells, but

in diverted segments it may become the first

energetic source [19].

Glutamine is usually absorbed in jejunum and

ileum. In this experiment, the small bowel segments

were maintained intact, ensuring the absorption of

the oral supplement of glutamine given to the

animals. This amino acid is equally metabolized if

absorbed from the intestinal lumen or from the

capillary bed, as it is expected to occur in colon

after colostomy [20–22].

Colostomy caused a significant (p<0.001) re-

duction of the crypts length and the supplement of

glutamine was able to avoid this change. Other

authors found a similar effect of deviation of fecal

stream on the crypts length [23]. Glutamine was

partially effective to avoid the reduction of the

partial volume of mucosa in CST–Gln group. The

mucosa volume remained at an intermediate value

between CST and control groups (p<0.05). A

previous article showed a reduction in partial

volume and mucosa weight in Wistar rats four

weeks after colostomy [12].

Diversion caused a significant (p<0.001)

increase of partial volume of muscularis mucosae

and the supplement of glutamine was not able to

avoid that change. Muscular tissue is rich on

glutamine-synthetase being able to provide large

amounts of glutamine and therefore being less

sensitive to variations of demand and delivery of

this aminoacid [24,25].

Partial volume of submucosa raised in CST

group when compared to control. Other authors

have studied the pathological changes of this layer

in diversion colitis in man and described an

increase in the conjunctive tissue and blood vessels

[26]. These changes can explain the increase in

volume. The use of glutamine was able to avoid this

change in CST–Gln group.

Partial volume of muscularis propria was dimi-

nished in both colostomized groups, and glutamine

was not able to avoid it, probably for the same reasons

discussed above on muscularis mucosae.

A high magnification study of mucosa showed a

fall on the partial volume of epithelium and a

raising of the partial volume of lamina propria

caused by colostomy. The supplement of L-glu-

tamine was able to avoid these changes and

promote a further raising on partial volume of the

epithelium, showing an important trophic effect of

this amino acid in epithelial cells and being in

agreement with findings of other authors studying

small bowel epithelium [8,9,27].

In conclusion, the oral supplement of L-glu-

tamine is able to avoid trophic changes on colon

mucosa and submucosa downwards colostomy.

These effects were not observed on muscular

layers. These findings can be useful either in the

prevention or in the treatment of patients suffering

from diversion colitis.

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