Direct determination of colonie nitric oxide level-A sensitive marker of disease activity in ulcerative colitis

Vol. 93, No. 3, 1998 ISSN OOQ2-9270/98/$19.00 1’11 SCOO2-9270(98)OOOO9-4

THE AMERlcAN JOURNAL OF GASTROENTEROLoGY Copyright 0 1998 by Am. COO. of Gastroenterology Published by Elsevier Science Inc.

Direct Determination of Colonic Nitric Oxide Level-A Sensitive Marker of Disease Activity in Ulcerative Colitis D. Rachmilewitz, Department

of Medicine,

M.D., R. Eliakim,

Hadassah

University

M.D., Z. Ackerman,

Hospital, Mount Jerusalem,

Objective: In active ulcerative colitis, colonic nitric oxide (NO) generation is enhanced and probably has an important role in its pathogenesis. We tested the reliability of an NO electrode in monitoring colonic NO levels in ulcerative colitis patients and control subjects and its possible usage as a marker of disease activity. Methods: Colonic NO level was determined by the NO detection system model NO-501 (InterMedical, Nagoya, Japan). The working electrode was inserted into a 7-mm diameter polyvinyl tube and introduced at a distance 6 cm from the anus. In each subject sigmoidoscopy was performed and mucosal biopsies were obtained. NO synthase (NOS) activity was determined by monitoring the conversion of 3H-arginine to citrulline. Results: Colonic NO level is significantly increased in patients with active ulcerative or Crohn’s colitismore than 2-fold higher than in control subjects. There was good correlation between colonic NO level and NOS activity and the clinical and endoscopic indices of disease activity. Conclusion: Direct determination of colonic NO level is convenient, and reliable, and may help to monitor disease activity in ulcerative colitis. (Am J Gastroenterol 1998;93: 409-412. 0 1998 by Am. Coll. of Gastroenterology)

INTRODUCTION Determination of the activity and severity of ulcerative colitis is difficult and, at present, far from satisfactory. Current activity indices are based on patients’ symptoms: number of bowel movements per day, presenceor absence of blood in the stool, patient’s condition overall, together with several objective parametersincluding body temperature, sedimentationrate, and hemoglobin level. A different approach relies on objective endoscopic findings that are less convenient to retrieve (1). We hereby propose a new and easy approach for the objective evaluation of disease activity in ulcerative colitis. The new approach is based on monitoring colonic levels of nitric oxide (NO). Received

Aug. 7, 1997; accepted

Oct. 20, 1997,

Scopus,

M.D., and F. Karmeli,

Hebrew

University

Hadassah

B.Sc Medical

School,

Israel

In addition to the involvement of eicosanoids, platelet activating factor, interleukins, and other cytokines in the pathogenesisof ulcerative colitis and Crohn’s disease(2), it has become apparent that NO is also an important mediator in its pathogenesis. Several methodological approaches established that in experimental colitis, as well as in ulcerative and Crohn’s colitis, colonic NO generation is enhanced and induced by stimulation of the enzyme responsible for its production, namely, nitric oxide synthase (NOS) (3). The methods used to determine the enhanced NO generation by the inflamed mucosa are determination of the generation of nitrites and nitrates, which are the end metabolites of NO (4.) determination of the actual level of citrulline, which is a byproduct of NO synthesis (5), monitoring the conversion of L-arginine to citrulline as an indicator of NOS activity (6), and direct measurement of colonic levels of NO (7). The direct measurement of colonic NO levels was based on determination of samples of colonic gas retrieved during colonoscopy by chemiluminescence. Its drawbacks are the necessity to subject patients to colonoscopy and the relative difficulty of measuring NO levels by chemiluminescence, which is dependent on an expensive instrument available only in sophisticated research laboratories. Monitoring NO levels with an NO electrode enablesthe easy detection and quantification of colonic NO levels and, therefore, may be used as a sensitive marker of diseaseactivity in ulcerative colitis patients. The present study reports its usage and correlation with the other available methods to determine colonic NO generation and to quantify diseaseactivity. MATERIALS

AND MET’HODS

Materials

Materials used included dithiothreitol, phenylmethylsulfonyl fluoride, EDTA, EGTA, NADPH, arginine, citrulline (Sigma Chemicals, Israel); Dowex AG50W-X8 (Na form) 100-200 mesh,and TRIS base(electrophoresisgrade) (BioRad, Richmond, CA); aquasol- (Dul?ont Co./NEN Research).

410

RACHMILEWITZ

et al.

AJG -- Vol. 93, No. 3, 1998 TABLE

1

Patients ’ Characteristics No. Normal control subjects Ulcerative colitis-active Ulcerative colitis-remission Crohn’s colitis-active

8 17 4 4

M:F 4:4 923 2:2 212

Patients Seventeen patients with active ulcerative colitis, four patients with active Crohn’s colitis, four patients with ulcerative colitis in remission, and eight control subjects were included in the study. Patient characteristics are detailed in Table 1. Patients with active ulcerative colitis had a clinical activity index of 9.3 and patients with ulcerative colitis in remission had a clinical activity index of 1.3, according to the index previously reported by us (1). Patients with active ulcerative colitis and Crohn’s colitis were treated with prednisone or with steroid enemas, and all patients were treated with a 5-ASA drug. In all patients with active ulcerative colitis sigmoidoscopy revealed active disease with an index of 8.5, according to the index previously reported by us (1). In patients with ulcerative colitis in remission the endoscopic index was 2.0, whereas in patients with active Crohn’s colitis sigmoidoscopy revealed ulcerated, friable, and inflamed mucosa from the anus to a distance of 540 cm. Control subjects were patients colonoscopied for various reasons (most of them for surveillance of colonic polyps), in whom no pathology was found in the colon. All subjects were examined either following bowel preparation for colonoscopy with 4 L of the precolonoscopic solution or following two Fleet enemas administered before sigmoidoscopy. In all of them, determination of colonic NO level was conducted before the fiberoptic procedure. During the procedure mucosal biopsies were obtained. Two biopsies were fixed for pathology and two to three biopsies were kept in 4°C 0.15 mol/L NaCl for immediate determination of NOS activity. Monitoring of colonic NO level We used the NO detection system model NO-501, InterMedical, Nagoya, Japan. The working electrode (made of platinum/iridium alloy coated membrane with a three-layered membraneconsisting of KCl, NO selective resin, and normal silicone membranes) and the counter electrode (made of carbon fiber) were inserted into a 7-mm diameter polyvinyl tube with holes in it. The reference carbon fiber electrode was positioned in the vinyl tube together with the working electrode, its tip projecting via one of its holes. The two electrodeswere positioned within 10 mm of each other to minimize the electrical noise, and care was taken to ensurethat they did not touch each other. The recordings of the NO electrode current were monitored on a Chronolog recorder. The electrode was calibrated for NO using S-

Age (yr) 35.0 24.9 36.0 38.0

k 2 2 k

10.2 2.4 5.0 5.1

Clinical

Activity 9.3 2 0.4 1.3 2 0.9

Index

Endoscopic

Index

8.5 2 0.4 2.0 2 1.1

nitroso-N-acetyl-DL-penicillamine (InterMedical, Nagoya, Japan), which serves as a stable standard NO generator, according to the method described by Ichimori et al. (8). The event in the electrode is linear with the NO concentration and the time constant is about 1.14 s, which allows almost real time in in vivo detection
AJG - March 1998

CONTROL

UC ACTNE

40

40

UC REMISSION

CROHN’S COLITIS ACTIVE

4i

*

30

0

0

** i

v 8

0

I-

ZE i

10

-

0

0

0

0

FIG. 1. Colonic NO levels. Mucosal NO levels were determined with an NO electrode in patients with ulcerative colitis, Crohn’s different subjects. Results are given as mean 5 SE. *Significantly different from normal subjects (p < 0.05). **Significantly ulcerative colitis (p < 0.05).

i 70 c; 7

l

colitis, and normal control from patients with active

.

/

R-o.64

FIG. 2. Correlation between colonic NO level and mucosal NOS activity. Colonic NO levels were determined with an NO electrode and, in the same subjects, NOS activity was determined with colonoscopic biopsies.

FIG. 3. Correlation between colonic NO level and clinical and endoscopic indices of disease activity. Colonic NO levels were determined with an NO electrode and, in the same subjects, clinical activity and endoscopic activity indices were determined according to methods described in Ref. 1. l = Endoscopic; 0 = Clinical.

RESULTS In patients with active ulcerative colitis colonic NO level determined with the NO microelectrode was found to be more than 2-fold higher than its level in normal subjects or in patients with disease in remission. In patients with active Crohn’s colitis, colonic NO level was also similar to that detected in patients with active ulcerative colitis (Fig. 1). There was a good correlation between mucosal NOS activity and colonic NO levels detected with the NO microelectrode (Fig. 2). There was also a very good correlation between colonic NO levels and the clinical and endoscopic indices of disease activity. (Fig. 3).

DISCUSSION The recently developed, selective NO microelectrodes enable real time monitoring of NO concentrations in vitro in cell lines and incubated tissues (9) in organs maintained ex viva (10) or in the living organism (11) In the present study, the selective NO microelectrode was used to monitor coionic mucosal NO levels in patients, with inflammatory bowel disease. Moreover, to verify the validity of the new electrode in monitoring mucosal NO levels, NOS activity was determined simultaneously. Monitoring colonic NO level with the NO electrode re-

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et al.

vealed, in patients with active ulcerative colitis and Crohn’s colitis, levels that were more than 2-fold higher than the levels detected in normal control subjects or in the colons of ulcerative colitis patients in remission. The validity of the NO electrode in detecting colonic NO levels is indicated by its good correlation with mucosal NOS activity determined by monitoring the conversion of L-arginine to citrulline. The latter method was previously used and showed enhanced colonic NOS activity in several models of experimental colitis, as well as in patients with active ulcerative colitis and Crohn’s colitis (3). Nitric oxide, being a free radical and, as such, a strong oxidant, may contribute to tissue damage in inflammation, either by itself or following its combination with O,-yielding peroxynitrite. Peroxynitrite was shown by us to induce severe colonic damage and inflammation following its intrarectal administration to rats (12). In active colitis, colonic NO generation was shown by several methods to be enhanced (3, 5, 7) and to derive from stimulation of NOS activity (3). Moreover, inhibition of NO generation by analogs of L-arginine was shown to ameliorate the extent and severity of several models of experimental colitis (13, 14). The microelectrode is simple and easy to use. Determination of colonic NO levels with this electrode is quick, reliable, and easy to perform. It is therefore suggested that colonic NO levels monitored by the NO electrode may be regarded as sensitive markers of disease activity in patients with active colitis. At present, in ulcerative colitis patients, a recommended approach for an objective evaluation of the disease severity is to have the patient undergo sigmoidoscopy and to have a direct look. This approach is awkward for patients, can be performed only after bowel cleaning, and is dependent on the subjective impressions of the observer. Direct monitoring of mucosal NO level with the NO microelectrode can be performed without previous bowel preparation, and expresses the disease activity in numbers that, as shown here, correlate with the clinical and sigmoidoscopic indices of disease activity. The main drawback of the method advised here as an objective index of the disease activity, is the relatively high cost of the monitor and the specific electrode. Direct monitoring of colonic NO level may also help to manage patients with severe refractory disease and, especially, patients with toxic megacolon. Nitric oxide, being an important neurotransmitter in the gut, has been suggested to be involved also in the pathogenesis of toxic megacolon. In experimental colitis, its levels were shown to correlate with the perimeter of the affected segment (15), whereas, in

AJG - Vol. 93, No. 3, 1998 patients with toxic megacolon, its mucosal and muscular levels were shown to be higher than in patients with active disease without megacolon (16). Medical treatment of toxic megacolon is difficult, and any objective parameter may help to improve the management of this dangerous complication. Reprint requests and correspondence: Daniel Rachmilewitz, M.D., Department of Medicine, Hadassah University Hoc,pital, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel.

REFERENCES 1. Rachmilewitz D. Coated mesalazine (5-amino-salicylic acid) versus sulphasalazine in the treatment of active ukerative colitis: A randomized trial. Br Med J 1989;298:82-6. 2. Eliakim R, Rachmilewitz D. Potential mediators in inflammatory bowel disease. Gastroenterol Int 1992;5:48 -56. 3. Rachmilewitz D, Stamler JS, Bachwich D, et al. Enhanced colonic nitric oxide generation and stimulated nitric oxide synthase activity in experimental colitis and in active inflamrratory bowel disease. Gut 1995;36:718-23. 4. Stuehr DJ, Marletta MA. Mammalian nrrite biosynthesis: Mouse macrophages produce nitrite and nitrate in response to Escherichia cob lipopolysaccharide. Proc Natl Acad Sci USA 1985:82:7738-42. 5. Middleton SJ, Shorthouse M, Hunter JO. Increased nitric oxide synthesis in ulcerative colitis. Lancet 1993;341:465-6. 6. Bush PA, Gonzalez NE, Griscavage JM, et al. Nitric oxide synthase from cerebellum catalyzes the formation cmf equimolar quantities of nitric oxide and citrulline from L-arginine. Biochem Biophys Res Commun 1992;185:960-6. 7. Lundberg JO, Hellstrom PM, Lundberg JM, et al. Greatly increased luminal nitric oxide in ulcerative colitis. Lancet 1994;344:1673-4. 8. Ichimori K, Ishida H, Fukabori M, et al. Fractical nitric oxide measurement employing an NO-selective electrode. Rev Sci Instrum 1994; 65:2714-8. 9. Miyoshi H, Nakaya Y. Endotoxin-induced non-endothelial nitric oxide activates the Ca*+-activated K’ channel in cultured vascular smooth muscle cells. J Mol Cell Cardiol 1994;26:1,$87-95. 10. Wang D, Hsu K, Hwang CP, et al. Measurement of nitric oxide release in the isolated perfused rat lung. Biochen Biophys Res Conunun 1995;208:1016-20. 11. Mitsuhata H, Saitoh J, Takeuchi H, et al. Production of nitric oxide in anaphylaxis in rabbits. Shock 1994;2:381-1.. 12. Rachmilewitz D, Stamler JS, Karmeli F, et al. Peroxynitrite induced rat . colitis-a new model ofcolonic inflammation. Gastroenterology 1993; 105:1681-8. 13. Rachmilewitz D, Karmeli F, Okon E, et al. Experimental colitis is ameliorated by inhibition of nitric oxide synthase activity. Gut 1995; 37x247-55. 14. Rachmilewitz D, Karmeli F, Okon E. Sulphydryl blocker-induced rat colonic inflammation is ameliorated by inhiition of nitric oxide synthase. Gastroenterology 1995;109:98-106. 15. Mourelle M, Guamer F, Vilaseca J, et al. Nitric oxide and toxic megacolon. In: Tytgat GNJ, Bartelsman JFWM, van Deventer SJH, eds. Inflammatory Bowel Diseases. London: Kluwer Academic Publishers, 1995:106-112. 16. Mourelle M, Casellas F, Guarner F, et al. Induction of nitric oxide syntbase in colonic smooth muscle from paients with toxic megacolon. Gastroenterology 1995;109: 1497-1502.