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APPLIED NUTRITIONAL INVESTIGATION Nutrition Vol. 14, No. 1, 1998
Gut Permeability, Intestinal Morphology, and
Nutritional Depletion
RENE
´R.W.J. VAN DER HULST,* MAARTEN F. VON MEYENFELDT,* BERNARD K. VAN KREEL,†
FREDERIK B.J.M. THUNNISSEN,‡ ROBERT-JAN M. BRUMMER,§ JAN-WILLEM ARENDS,‡
AND PETER B. SOETERS*
From the *Department of Surgery, †Department of Clinical Chemistry, ‡Department of Pathology, and
§Department of Gastroenterology, University of Limburg, Maastricht, The Netherlands
Date accepted: 9 April 1997
ABSTRACT
Nutritional depletion increases the risk for postoperative complications. The intestinal barrier may be important in the
underlying pathophysiologic mechanism. In this study, 26 patients were evaluated to determine whether nutritional depletion was
related to gut integrity and intestinal morphology. Nutritional depletion was estimated by calculating percentage ideal body
weight (PIB) or percentage ideal fat free mass (PIFFM). To assess gut integrity, a lactulose/mannitol (L/M) test was performed.
Duodenal biopsies were taken, and villous height, crypt depth, number of IgA-producing plasma cells, intraepithelial lympho-
cytes (IELs), and proliferating index were determined. The L/M ratio was increased, and villous height was decreased in depleted
patients. Depletion was not associated with differences in the number of immune cells or proliferating index. The number of
IgA-producing plasma cells was positively correlated with the L/M ratio. This study shows that nutritional depletion is associated
with increased intestinal permeability and a decrease in villous height. Nutrition 1998;14:1–6. ©Elsevier Science Inc. 1998
Key words: nutrition, intestine, depletion, permeability, lactulose-mannitol
INTRODUCTION
The association between nutritional depletion, septic compli-
cations,
1,2
and increased mortality rates
3
in postoperative patients
is well established. The exact underlying mechanism is not
known.
It has been hypothesized that the gut plays an important role in
the development of complications in the postoperative patient.
4
An important function of the healthy gut is to prevent bacteria and
endotoxins from reaching the portal circulation. This physiologic
barrier is maintained by the mucous layer, the epithelial cells with
their tight junctions, and the gut-associated lymphoid tissue
(GALT). Impairment of one or several components of this intes-
tinal barrier may result in bacterial translocation or endotox-
emia.
5,6
Glutamine, a conditional essential amino acid, is used as fuel
for rapidly dividing cells, for example, enterocytes and lympho-
cytes.
7,8
A diminished glutamine supply during parenteral nutri-
tion and enteral starvation results in morphologic changes and
increased intestinal permeability.
9
Recently, we showed that nu-
tritional depletion is associated with decreased concentrations of
glutamine in the intestinal mucosa.
10
Nutritional depletion, via
decreased glutamine supply, may impair intestinal barrier func-
tion.
In animal experiments, protein malnutrition results in an in-
creased risk of endotoxin-related bacterial translocation and in-
creased susceptibility to the lethal effects of endotoxins.
11,12
The
purpose of this study was to investigate the potential relationship
between nutritional depletion, intestinal morphology, and perme-
ability in humans.
METHODS
Patients
Metabolically stable patients (temperature between 36.5 and
38°C, no intraabdominal abscesses, no signs of respiratory or
cardiac failure) between 18 and 80 years of age admitted to the
nutritional support team were eligible to enter the study. All
patients were admitted because they were not allowed or were
unable to receive enteral nutrition. The study was performed
before parenteral nutrition was initiated. Patients with renal or
liver failure, diabetes mellitus, ileus, or congenital metabolic dis-
orders, and patients who received parenteral nutrition within 3
weeks preceding the study were excluded. For control values of
Correspondence to: R.R.W.J. van der Hulst, Department of Surgery, University of Limburg, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.
Nutrition 14:1–6, 1998
©Elsevier Science Inc. 1998 0899-9007/98/$19.00
Printed in the USA. All rights reserved. PII S0899-9007(97)00385-7
the lactulose/mannitol (L/M) test, a control group of 12 healthy
persons was studied. Patients were fasted overnight and were
studied in the morning. Blood was collected for routine biochem-
ical indices, total protein, albumin, and prealbumin.
Percentage of ideal body weight (PIB) was calculated using the
Metropolitan Life Insurance Company tables 1983.
13
In 20 pa-
tients, a bioimpedance measurement was performed to estimate
fat-free mass using a standard formula.
14
The percentage of ideal
fat free mass (PIFFM) was calculated assuming an ideal fat-free
mass to be, respectively, 88% and 78% of the ideal body weight
for men and women. Patients were classified nutritionally depleted
if their PIFFM was less than 90% or, in case no PIFFM was
calculated (6 patients), if their PIB was less than 90%.
The study was approved by the Medical Ethical Committee of
the University Hospital Maastricht. Written informed consent was
obtained from every patient.
Permeability Tests
After a duodenoscopy during which biopsies were taken, a
catheter was positioned in the duodenal bulb and a solution
containing 10 g of lactulose, 0.5 g of mannitol, and5gofD-xylose
in 65 mL of water (osmolality 51474 mOsm/kg) was infused
during 1 min. Patients had to empty their bladder before the
endoscopy and were asked to collect all urine voided during the
next 6 h. The collected urine was preserved with 5 mL thymol
10%. Control values for lactulose/mannitol/xylose absorption
were obtained from 12 healthy volunteers without any evidence of
systemic or gastrointestinal disease. In the control group, the
solution was given orally. Urinary lactulose and mannitol were
determined by gas-liquid chromatography.
15
Xylose was mea-
sured enzymatically.
16
Excretion percentages and L/M ratio were
determined as previously described.
15
Intestinal Histology
Intestinal biopsies were taken from the second part of the
duodenum distal to the level of the hepatopancreatic ampulla.
Specimens of the duodenum were immediately fixed either in
ethanol (two specimens for staining of proliferative activity) or
in Bouin’s solution (two specimens for villous and crypt mea-
surement and immunohistochemical staining of plasma cells
and lymphocytes). Fixed tissues were then carefully oriented
and embedded in paraffin. For each staining, at least six well-
oriented sections of 4
m
m thickness were obtained at different
levels of the specimen. Quantitative measurements were per-
formed by one of the authors (R.V.D.H.) with coded sections,
without knowledge regarding the identity of the corresponding
patients. For ethical reasons, control biopsies were not ob-
tained. For morphometry, sections were stained with hematox-
ylin and eosin. Morphometry measurements for villous height
and crypt depth were performed using an automatic inter-
active analysis system (Jandel video analysis system, Erkath,
Germany) as has been described recently.
9
Proliferating duodenal crypt cells were detected using immu-
nostaining for proliferating cellular nuclear antigen (PCNA). Tis-
sue sections were deparaffinized with xylol and washed with
ethanol 100%. Endogenous peroxidase activity was blocked with
H
2
O
2
0.6% in methanol for 15 min. Subsequently, sections were
incubated with target unmasking fluid (TUF; Monosan, The
Netherlands) 10 min at 90°C, washed with TRIS buffered saline
(TBS) and incubated with 1:300 diluted antibody to PCNA (PC10,
Dako, Withoorn, The Netherlands M879) for 60 min at 37°C,
followed by washing in TBS and incubation with peroxidase-
labeled rabbit antimouse 1:200 (P270, Dako). Immunoreactivity
was demonstrated using DAB (3,3 diaminobenzidine D-5637;
Sigma Chemical Co., St. Louis, MO, USA), and sections were
counterstained with hematoxylin-eosin. The PCNA labeling index
was obtained by dividing the number of positive PCNA cells in
crypts by the total number of enterocytes in the crypt multiplied
with 100. The progressive mean was determined for PCNA in at
least 30 subsequent crypts. A stable pattern was obtained after
measuring 10 crypts. Therefore, PCNA labeling index was ob-
tained after counting 10 crypts as described by Sarraf et al.
17
The
intraobserver and interobserver coefficient of variation (CV) for
PCNA were 3% and 5%, respectively.
For IgA and CD3 immunostaining, the same blocking and
hydration procedure was used. Sections were incubated with an
antibody to IgA (Rabbit antihuman IgA, A 408 Dako), or an
antibody to CD3 (Rabbit antihuman T-Cell CD3, A 452 Dako)
for 45 min at room temperature. After washing with TBS,
peroxidase-labeled swine antirabbit (P217, Dako, The
Netherlands) and DAB was applied. Plasma cells were counted
in the lamina propria and expressed as the number of cells per
high power field as described by Alverdy et al.
18
At least 10
high power fields were counted. A correction was made for the
surface of lamina propria using a transparent test grid overlay
with a random dot design as described by Aherne and
Dunnill.
19
Quantitative measurement of CD3-positive cells in
the epithelium was obtained by counting lymphocytes present
in 10 villi or in relation with 1000 enterocytes. Intraepithelial
lymphocytes (IELs) presence was expressed as the number of
lymphocytes counted per 100 enterocytes.
20
The quantification
procedure of IEL and IgA cells was similar as the procedure for
PCNA counting. Because the fraction of IgA and CD3 express-
ing cells was higher than the number of PCNA-expressing
cells, theoretically a similar or lower CV and progressive mean
is to be expected. Therefore IEL and IgA counting was per-
formed in, respectively, 10 villi and 10 high power fields.
Calculations and Statistics
Results are presented as means 6SEM. Levels of significance
were set at P,0.05. Group comparison for statistical significance
was performed using the Mann-Whitney U test. The Pearson test
was used for correlation analysis. The statistical procedures were
performed with a SPSS-PC1software program (SPSS, Inc.,
Chicago, IL, USA).
RESULTS
Patients
Intestinal biopsies were taken in 26 patients submitted to the
care of the nutritional support team. Intestinal permeability was
studied in 23 of these patients. Patients were treated for inflam-
matory bowel disease (IBD; n515), cancer (Ca; n56), and
other diagnoses (NoCa/NoIBD) with subileus (n51), pancreatitis
(n51), pyloric stenosis (n51), or fistula (n52). Patient data
are summarized in Table I. Fourteen patients were considered
nutritionally depleted because they had a PIFFM of less than 90%
(8 patients) or a PIB less than 90% (6 patients). The L/M control
group consisted of 6 men and 6 women with a mean age of 31 6
3 yrs and weight of 70 65 kg.
Permeability Tests
No significant differences in L/M ratio nor in absolute excre-
tion percentages between the different patient groups were ob-
served (Table II). All patients had higher L/M ratios compared
with controls (Fig. 1). Patients who were considered to be nutri-
tionally depleted did have a higher L/M ratio than patients who
were not nutritionally depleted (P,0.05; Fig. 1). Excretion
percentages of lactulose, mannitol, and xylose in depleted patients
were not significantly different from those in nondepleted patients
(Table III). The L/M ratio was correlated with the number of IgA
plasma cells in the lamina propria (Fig. 2). No correlation was
GUT PERMEABILITY AND NUTRITIONAL DEPLETION2
observed between L/M ratio and the number of IELs, villous
height, crypt depth or proliferative activity.
Intestinal Morphology
There were no significant differences in morphologic param-
eters between the three patient categories (Table II). The PIB and
PIFFM were both positively correlated to villous height (Fig. 3);
nutritional depletion was associated with a decrease in villous
height. Villous height in the depleted group seemed to be lower
compared with a recently described healthy control group.
9
No
significant differences were observed between depleted and non-
depleted patients regarding crypt depth, plasma cells, proliferative
activity, or percentage of intraepithelial lymphocytes (Table IV).
These parameters were all within the ranges given in the litera-
ture.
9,17,21–24
The decrease in villous height was not associated
with a decrease in proliferative activity. To the contrary, with
decreasing villous height, proliferative activity was significantly
increased (r520.42, P50.03) (Fig. 4).
DISCUSSION
This study was performed to assess the relationship between
nutritional depletion, intestinal permeability, and morphology.
The association between nutritional depletion and intestinal per-
TABLE I.
PATIENT CLINICAL PARAMETERS
Non-
depleted Depleted
Patient data
Sex (male/female) 9/3 7/7
Diagnosis (n)
IBD 7 7
Cancer 2 4
Fistula 1 1
Gastric stenosis 1 —
Diverticulitis — 1
Pancreatitis 1 —
Subileus — 1
Medication
Sulfasalazine (n) 5 8
Corticosteroids (n) 5 4
Laboratory results of blood
Total protein (g/L) 65 (1) 62 (2)
Albumin (g/L) 34 (2) 29 (2)
Prealbumin (g/L) 0.2 (0.02) 0.1 (0.02)*
Leucocytes (10
9
/L) 9.8 (0.8) 8.7 (0.8)
Thrombocytes (10
9
/L) 372 (45) 389 (46)
ESR (mm/h) 26 (7) 43 (8)
Hb (mmol/L) 7.7 (0.3) 7.5 (0.3)
Values presented as means (SEM).
*P,0.05, Mann-Whitney.
IBD, inflammatory bowel disease; ESR, erythrocyte sedimentation rate;
Hb, hemoglobin.
TABLE II.
DIAGNOSTIC GROUP IN RELATION TO LACTULOSE/MANNITOL PERMEABILITY AND MORPHOLOGY
IBD
(n514) Cancer
(n55) NoCa/NoIBD
(n54)
L/M ratio 0.14 (0.03) 0.14 (0.04) 0.10 (0.06)
Lactulose % 1.7 (0.4) 1.6 (1.0) 0.9 (0.8)
Mannitol % 13.3 (2.6) 12.7 (6.7) 8.4 (2.8)
Xylose % 21.7 (3.1) 18.5 (6.0) 12.9 (8.0)
Villous height (
m
m) 472 (16) 416 (31) 446 (34)
Crypt depth (
m
m) 162 (12) 185 (20) 148 (7)
IEL (n/100 enterocytes) 28 (2) 21 (4) 27 (4)
IgA (n/HPF) 65 (3) 76 (9) 61 (7)
PCNA (LI) 28 (2) 34 (5) 30 (4)
L/M, lactulose/mannitol; IBD, inflammatory bowel disease; IEL, intraepithelial lymphocytes; PCNA, proliferative cellular nuclear antigen; HPF, high
power field.
Values presented as means (SEM). No significant differences were observed between the three different groups of patients.
FIG. 1. Nutritional depletion and lactulose/mannitol permeability. Nutri-
tional depletion was associated with an increased intestinal permeability.
Control versus depleted and nondepleted P,0.05. Depleted versus
nondepleted P,0.05.
GUT PERMEABILITY AND NUTRITIONAL DEPLETION 3
meability in humans was first described by Maxton et al.
25
Star-
vation in obese patients and nutritional depletion in patients with-
out gastrointestinal disease receiving enteral nutrition were
associated with increased permeability. In our study, intestinal
permeability and intestinal morphology were assessed in nutrition-
ally depleted patients who were unable or not allowed to receive
enteral nutrition. Before parenteral nutrition was initiated, intestinal
permeability was measured and duodenal biopsies were obtained and
related to the degree of depletion. For ethical reasons, duodenal
biopsies were not taken in the control group and permeability in the
control group was measured by giving the test solution orally. This
may have biased the differences in permeability between patients and
controls. In the control group, the solution was diluted by the gastric
juice before entering the duodenum. In the patient group, the solution
was infused undiluted into the duodenal bulb. The hyper-
osmotic solution used may have accentuated increased permeability
in patients with minimal villous atrophy or may have increased
permeability even in the normal intestine.
26,27
In addition, the effect
of taking biopsies on intestinal permeability measurements is un-
known. Therefore, differences between controls and patients may be
confounded by the use of hyperosmotic stress and the taking of
biopsies. However, the differences in permeability between controls
and patients with IBD were comparable to data previously pub-
lished.
28
Furthermore, the main purpose of this study was not to
compare patients with controls but to compare depleted and nonde-
pleted patients in which the method of permeability measurement
was the same. Permeability proved to be clearly increased in the
nutritionally depleted patient group as compared with the nonde-
pleted group.
The ratio between crypts and villi is often used to describe
changes in intestinal morphology, for example, celiac disease.
Because in animal models of nutritional depletion, both villous
height and crypt depth are decreased
29
and changes in villous
height and crypt depth may disappear when calculating a villous:
crypt ratio. Therefore, in this study, crypt depth and villous height
were presented separately. The decrease of villous height in nu-
tritionally depleted patients is in line with experimental observa-
tions in chronic malnourished rats.
29
A study on the effect of
alcohol on mucosa morphology in humans showed weight param-
eters to relate to mucosa morphology, supporting our observa-
tions.
30
Assuming that PCNA-positive cells traverse normally
through the cell cycle, the observation that proliferative activity is
increased associated with a reduced villous height suggests that
the enterocytes have a decreased cell life.
Nutritional depletion results in increased bacterial translocation in
animal experiments.
11
The gut barrier is of critical importance in the
prevention of translocation of bacteria and their products into the
portal circulation and the mesenteric lymph nodes. Although trans-
location of endotoxins through an intact intestinal mucosa does not
occur in humans,
31
the concept of translocation may be complex and
FIG. 2. Correlation between IgA plasma cells in the lamina propria and
permeability. An increase in permeability was associated with an increase
in number of IgA producing plasma cells in the lamina propria of the
duodenum.
FIG. 3. Correlation between villous height and nutritional depletion.
Percentage ideal body weight (PIB) and percentage of ideal fat-free mass
(PIFFM) were correlated with villous height in the duodenum.
TABLE IV.
NUTRITIONAL DEPLETION AND MUCOSAL MORPHOLOGY
Depleted Non-
depleted
Villous height (
m
m) 419 (17) 491 (6)*
Crypt depth (
m
m) 165 (13) 166 (11)
IEL (n/100 enterocytes) 27 (3) 24 (2)
IgA (n/HPF) 64 (4) 70 (5)
PCNA (LI) 32 (3) 27 (2)
IEL, intraepithelial lymphocytes; PCNA, proliferative cellular nuclear
antigen; HPF, high power field.
Laboratory values presented as means (SEM). * P,0.01.
TABLE III.
NUTRITIONAL DEPLETION AND URINARY EXCRETION
Control
(n512) Depleted
(n513) Non-depleted
(n510)
Lactulose % 0.5 (0.1)* 2.0 (0.5) 0.9 (0.3)
Mannitol % 19.2 (2.6)* 12.9 (3.5) 11.5 (1.6)
Xylose % 29.9 (1.8)* 20.6 (3.4) 18.1 (4.2)
Values presented as means (SEM).
*P,0.05 versus depleted and non-depleted.
GUT PERMEABILITY AND NUTRITIONAL DEPLETION4
must be studied in relation to intestinal morphology and factors
causing changes in intestinal morphology. Several factors contribute
to the intestinal barrier function. The first factor is the mucous layer.
Elimination of the mucous layer results in a notable increase in the
number of bacteria directly adherent to the enteric surface and an
increased dissemination of normal intestinal bacteria to extraintestinal
tissues such as the liver and spleen.
5
The second part of the intestinal
barrier is the epithelium itself, consisting of enterocytes, mucus-
producing goblet cells, amine precursor uptake and decarboxylation
(APUD) cells, and IELs. The epithelial cells are bound by so-called
tight junctions in the zona occludens.
32
The gut-associated lymphoid
tissue forms the third part of the intestinal barrier. It is composed of
immune cells in the lamina propria (plasma cells, lymphocytes,
macrophages, and eosinophils), aggregates of lymphocytes called
Peyer’s patches, and the cells in the mesenteric lymph nodes.
5
The
dual sugar only tests the epithelium part of the intestinal barrier.
Theoretically, an increase in L/M permeability may be caused by a
decrease in mannitol absorption and/or an increase in lactulose ab-
sorption. Decreased mannitol absorption is a result of a diminished
absorptive area.
33
Increased permeation of lactulose may in theory be
due to a facilitated diffusion of lactulose into the crypt region as a
consequence of decreased villous height. Because the tight junctions
in the crypt region are more permeable, this will result in an increased
diffusion of lactulose.
34
The morphologic data suggest that a decrease in villous height
is at least partially responsible for the changes in L/M ratio
between the nondepleted and depleted patients. The data on man-
nitol and xylose absorption, however, do not prove that there is a
difference in absorption between the nondepleted and depleted
patients, because there was no difference in mannitol and xylose
excretion between these two groups. In contrast, lactulose excre-
tion was higher in the depleted patients. Therefore, it seems that
the decreased villous height results in a facilitated diffusion of
lactulose into the crypt area, resulting in increased lactulose per-
meability and increased L/M ratio. The comparable xylose excre-
tion between nondepleted and depleted patients suggests that the
development of nutritional depletion is not the result of changes in
absorption and that the observed differences in permeability be-
tween the patient groups are secondary to nutritional depletion and
are not causing nutritional depletion. Without doubt, it remains
difficult to separate cause and effect when studying nutritional
depletion in relation to intestinal permeability. Our data add fur-
ther support to the findings of Maxton et al.,
25
who showed that
intestinal permeability increased as a result of starvation.
Another explanation for an increased L/M ratio may be an
increased lactulose permeation due to loosening of the tight junc-
tions.
33
Tight junctions are permeable to small molecules. They
constitute a dynamic complex that is involved in the regulation of
nutrient uptake.
32
In disease, loosening of the tight junctions is
thought to be a result of production of oxygen radicals by neutro-
phils invading the epithelium.
35,36
Activation of neutrophils ap-
pears to be an important factor in the pathogenesis of tissue
injury.
37
In the gut, neutrophils may become activated by endo-
toxins absorbed by the gut. There is little evidence that there is
translocation of endotoxins in the intact gut. Endotoxin uptake,
however, occurs in the diseased intestine.
31
Also, endotoxin in-
creases intestinal permeability in humans.
38
In addition, intestinal
permeability is increased in critically ill patients.
39
Neutrophils
may also become activated by the production of interferon-
g
by
intraepithelial lymphocytes.
40
Intraepithelial lymphocytes are the
first cells of the immune system that are exposed to potentially
pathogenic organisms and play a key role in gut barrier function.
41
In addition to neutrophil activation, interferon-
g
has also a direct
effect on the loosening of tight junctions.
42
In this study, the number of intraepithelial lymphocytes was
not significantly related to permeability. In animals, the number of
immune cells in the epithelium as well as in the lamina propria of
the small bowel decrease during pure nutritional depletion.
43,44
In
clinical depletion and in malnutrition in Third World countries,
however, comparable amounts of cells or even increased numbers
of immune cells are found in the intestine.
45
This may be the result
of activation of gut immune cells possibly caused by translocation
of bacteria or their products. The activated intraepithelial lympho-
cytes may subsequently directly or indirectly increase epithelial
permeability. The relation between plasma IgA cells and L/M ratio
is indicative for an activation of the gut-associated immune cells.
One study
46
of patients with primary IgA deficiency did not show
increased permeability in these patients. The plasma IgA cells
therefore do not seem to regulate intestinal permeability directly.
The increased number of IgA cells in patients with increased
permeability must indicate loss of gut barrier function against
immune stimulating agents and shows that the L/M test reflects
not only sugar permeation in these patients.
The difference in permeability between the controls and non-
depleted patients is mainly caused by a decreased mannitol ab-
sorption in the patient population (Table III). A small increase in
lactulose excretion was also observed in the patient group. This
may be caused by the difference in osmolarity of the solutions
given in the control and patient group. Although mannitol absorp-
tion was decreased in the nondepleted patient group villous height
was in the normal range.
9
A possible explanation for this obser-
vation could be that the changes causing decreased mannitol
absorption in the patients are located distal from the duodenum
where the intestinal biopsies were taken.
In conclusion, nutritional depletion results in increased ‘‘epithelial
permeability,’’ probably caused by a facilitated diffusion of macro-
molecules into the crypt area as a result of decreased villous height
but also possibly as a result of opening of tight junctions. In addition,
increased L/M permeability in nutritional depletion is associated with
an increased number of IgA plasma cells in the lamina propria.
ACKNOWLEDGMENTS
The authors thank Margriet Pijls, Anniek Moors, Janine
Hoefnagels, and Margriet Rouflart for their help with this study.
FIG. 4. Correlation between villous height and proliferating cellular nu-
clear antigen (PCNA). Villous height and PCNA were slightly negatively
correlated.
GUT PERMEABILITY AND NUTRITIONAL DEPLETION 5
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GUT PERMEABILITY AND NUTRITIONAL DEPLETION6
