COLONIC PROTEINASES IN ULCERATIVE COLITIS
COLONIC PROTEINASES: INCREASED ACTIVITY IN PATIENTS WITH
DANIEL BUSTOS, GUSTAVO
NEGRI, JUAN ANDRES DE PAULA, MARIA DI CARLO, VIVIANA YAPUR, ANDREA
FACENTE, ALDO DE PAULA
Gastroenterología y Enzimología Clínica, Departamento de
Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Universidad
de Buenos Aires; Servicio de Gastroenterología, Hospital Italiano,
Key words: ulcerative colitis, proteinases, neutrophil
to study the colonic intraluminal proteinase-antiproteinase imbalance
under inflammatory con- ditions, we determined proteolytic activity
(PA), alpha-1-antitrypsin and the activities of trypsin, chymotrypsin
and neutrophil elastase in feces from patients with ulcerative colitis
(UC) comparing the results with a control group. A fecal sample was
obtained from each of 25 patients with ulcerative colitis and 10
control subjects were studied. The severity of the disease was
assessed by the Truelove index. Proteolytic activity was measured
using azocasein as proteolytic substrate. The fecal concentration of
alpha-1-antitrypsin was measured by radial immunodiffusion and the
activities of the enzymes were measured using specific substrates. We
found an increase in fecal PA, alpha-1-antitrypsin and neutrophil
elastase in patients with UC and the correlation between the severity
of the disease and the PA was statistically significant (r = 0.62, P
< 0.05). We conclude that elevated colonic proteinase activity
could contribute to the pathophysiology of ulcerative colitis.
intracolónicas: actividad incrementada en pacientes con colitis
ulcerosa. Con el obje- tivo de estudiar el posible desbalance del
sistema proteasa-antiproteasa en el medio intraluminal colónico bajo
condiciones inflamatorias, se determinó la actividad proteolítica
(AP), la concentración de alfa-l-antitripsina y las actividades de
tripsina, quimotripsina y elastasa de neutrófilos en heces de
pacientes con colitis ulcerosa (CU), comparando los resultados con un
grupo control. La severidad de la enfermedad en los pacientes con CU
fue determinada por el índice de Truelove. La AP fue medida usando
azocase?a como sustrato, la concentraci? fecal de alfa-1-antitripsina
por inmunodifusi? radial y las actividades enzim?icas usando sustratos
espec?icos. Se observ·un incremento en la AP, alfa-1-antitripsina y
elastasa de neutr?ilos en heces de pacientes con CU y la correlaci?
entre la severidad de la enfermedad y la AP fue estad?ticamente
significativa (r = 0.62, p < 0.05). Concluimos que la elevaci? de
la actividad de proteasas intracol?icas podr? estar relacionada a la
fisiopatología de la colitis ulcerosa.
Postal address: Dr. Daniel Bustos, Departamento de
Bioquímica Clínica, Facultad de Farmacia y Bioquímica, UBA, Junín
956, 1113 Buenos Aires, Argentina
Received: 4-XI-1997 Accepted: 8-V-1998
Under normal conditions intraluminal colonic proteolytic activity
(PA) is the combination of PA from pancreatic enzymes that reach the
large intestine and PA from colonic microflora1-4. In patients with
ulcerative colitis (UC) proteinases from neutrophils may contribute to
intraluminal proteolysis5. Proteinases in the gastro-intestinal lumen
can break the mucus barrier, digest the underlying epithelium and
produce mucosal damage. Neutrophil elastase liberated into the
intraluminal milieu can be neutralized by alpha-1-antitrypsin, thus
protecting the colonic mucosa from proteolysis5. In the oxidative
burst of neutrophils, myeloperoxidase (MPO) plays a major role by
catalysing the reaction between hydrogen peroxide and chloride ions,
to produce the highly cytotoxic hypochlorous acid6. Both MPO and
reactive oxygen species may produce inactivation of
alpha-1-antitrypsin7, leading to an increase in the colonic PA. In
orden to study the colonic intraluminal proteinase-antiproteinase
imbalance under inflammatory conditions, we measured PA,
alpha-1-antitrypsin, and the activities of trypsin, chymotrypsin and
neutrophil elastase in feces from patients with ulcerative colitis
comparing the results with a control group.
Materials and Methods
A fecal sample was obtained from each of 25 patients with
ulcerative colitis, and 10 control subjects (members of the laboratory
staff) were studied. Spontaneous feces were collected and the samples
were kept at -20°C until analysed. The severity of the disease was
assessed by the Truelove index8. Five hundred milligrams from each
fecal sample were homogenized in 5 ml of 0.05 mol/L tris buffer, pH
7.4. After centrifugation at 3.000 g for 10 min at 4 °C the
supernatant was assayed for trypsin, chymotrypsin and neutrophil
Chymotrypsin activity was measured using 0.1 mM of
succinyl-Ala-Ala-Pro-Phe 4 nitroanilide (Sigma) as substrate9. The
assay were performed at 25 °C in 0.1 M tris-HCl and 0.01 M CaCl2, pH
7.8. For trypsin activity, we used a 0.01 M solution of
benzoyl-DL-arginine P-nitroanilide (Sigma) as substrate10. The assays
were performed at 25 °C in 0.05 M tris buffer, pH 8.2, containing
0.02 M CaCl2. For elastase activity a solution of
Methoxysuccinyl-L-alanyl-L-prolyl-L-valine-p-nitroanilide was used11.
The cuvette contained 0.32 mM of substrate,and 20 uL of elastase
sample, in phosphate-buffered saline at pH 7.4.
For measurement of proteolytic activity and alpha-l-antitrypsin
concentration 1 gr of feces was homogenized in 0.1 M sodium phosphate
buffer, pH 7.0, to produce 10% (W/V) slurries. The proteolytic
activity were carried out as described1 using azocasein as proteolytic
substrate. The fecal concen-tration of alpha-1-antitrypsin was
measured by radial immu-nodiffusion12.
The Wilcoxon signed rank test and the Kruskal-Wallis test were
employed for paired samples and independent samples, respectively.
Correlations were assessed with the Spearman rank correlation test.
As table 1 shows, the PA in patients with ulcerative colitis was
higher than in healthy subjects (p < 0.01). The Spearman
correlation coefficient between PA and severity of the disease was
statistically significant, 0.62 (P < 0.05). As written in table 2,
we found and increased fecal activity of neutrophil elastase in
patients with ulcerative colitis. The activity of this enzyme was
undetectable in healthy subjects, but it was present in twenty-three
out of 25 patients with ulcerative colitis. We did not find a
significant correlation between the activity of this enzyme and the
severity of the disease. Despicted in Table 3 is the concentration of
alpha-l-antitrypsin in the supernatant of fecal homogenates from
patients with ulcerative colitis and normal subjects. We found an
increased fecal concentration of alpha-1-antitrypsin in patients with
ulcerative colitis. However we found no significant correlation
between the severity of the disease and the fecal concentration of
This study shows that fecal proteolytic activity was significantly
higher in patients with ulcerative colitis than in healthy subjects
and this PA had a significantly positive correlation with the severity
of the disease.
The increased fecal neutrophil elastase in patients with UC suggests
that this enzyme may have contributed to the increased fecal
proteolytic activity in these patients. However, since there was no
correlation between the level of the elastase and the severity of the
disease, other proteolytic enzymes of white cell origin, such as
collagenase and gelatinase, may have played a part13.
Intracolonic neutrophil elastase could contribute to the
pathophysiology of ulcerative colitis by a direct effect on colonic
epithelial cells or by liberating a mediator of the inflammation. In
this regard it has been shown that neutrophil elastase stimulates
platelet activating factor (PAF) synthesis by PMN and endothelial
cells to levels comparable with those observed after the addition of
tumor necrosis factor (TNF) or after phagocytosis14. PAF shows a wide
range of biological effects inducing aggregation of platelets15,
promoting chemotaxis of neutrophils and monocytes16, 17, increasing
vascular permeability and altering the vascular tone18. We also found
an increased fecal concentration of alpha-1-antitrypsin in patients
with ulcerative colitis. Similar results have been shown by others19,
20. This fecal elevation probably reflects an increased permeability
of the mucosal barrier, seen as a result of the inflammatory process
in which plasma proteins diffuse into the intraluminal space of the
intestinal tract. We found an increased activity of fecal neutrophil
elastase in patients with ulcerative colitis, so that
alpha-l-antitrypsin was unable to produce a total inhibition of this
enzyme. Alpha-1-antitrypsin appears to be overcome by large amounts of
free neutrophil elastase. The other possibility is that the catalytic
activity of myeloperoxidase with hydrogen peroxide and Cl gives rise
to highly reactive oxigen species, including O2 and HOCl wich can
oxidize the alpha-1-antitrypsin, especially to methionine in the
active center of the molecule. After oxidation, alpha-1-antitrypsin
has a much lower affinity to elastase21. In conclusion, we found an
increased PA in feces from patients with ulcerative colitis and this
PA had a significant correlation with the severity of the disease. It
is probably due to a protease-antiprotease imbalance. Further studies
are necessary to evaluate whether these patients profit from already
available therapeutic options, such as infusion of active
Acknowledgement: Ths work was supported in part by a grant
from University of Buenos Aires.
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TABLE 1.– Proteolytic activity (mg Azocasein/h/gr of feces) in
patients with ulcerative colitis and normal subjects
Control subjects Ulcerative colitis
(n = 10) (n = 25)
Median 2.88 10.80*
Range (0.70-3.60) (1.09-25.85)
Quiescent Mild Moderate Severe
(n = 6) (n = 8) (n = 7) (n = 4)
Median 1.30 7.60 18.20 20.6
Range (1.09-6.01) (1.27-18.22) (4.86-25.85) (16.5-32.4)
* p < 0.01
TABLE 3.– Concentration of Alpha-1-antitrypsin (mg/dl) in
patients with ulcerative colitis and healthy subjects
Controls Ulcerative colitis
(n = 10) (n = 25)
Median 1.7 48.0*
Range (0-3.8) (6.5-117.0)
Quiescent Mild Moderate Sever
(n = 6) (n = 8) (n = 7) (n = 4)
Median 10.5 66.5 30.3 51.0
Range (6.5-31.5) (18.0-117.0) (6.5-70.0) (29.0-74.0)
* p < 0.01
TABLE 2.– Trypsin, chymotrypsin and neutrophil elastase in
patients with ulcerative colitis and control subjects
Trypsin Chymotrypsin Neutrophil elastase
Control 19.7 12.6 0
(n = 10) (16.9-81.7) (2.2-44.8) (0-0)
Ulcerative 25.1 23.3 3.48*
colitis (3.8-55.3) (5.50-118.4) (0-27.88)
(n = 25)
* p < 0.01. The results are expressed as median and range of