Article Text

Excess nitric oxide in ulcerative colitis may be generated by nitric oxide synthase independent pathways
  1. Z ZHANG,
  3. E CARTY,
  1. MRC Collaborative Centre,
  2. 1–3 Burtonhole Lane,
  3. Mill Hill, London NW7 1AD, UK and
  4. Inflammation Research Group and
  5. Digestive Diseases Research Centre,
  6. St Bartholomew’s and Royal London
  7. School of Medicine and Dentistry,
  8. London E1 2AD, UK

Statistics from

Editor,—Kimura et al(Gut1998;42:180–7) provide further data in support of the proposal that production of nitric oxide (NO) in ulcerative colitis (UC) is ascribable primarily to the action of inducible NO synthase (NOS) on l-arginine.1-3

This reaction relies on molecular oxygen as a substrate (fig1).4 However, the Po 2 of gas in the normal colon is low (sometimes only 1–2 mm Hg), not only as a result of bacterial utilisation of oxygen, but also reflecting mucosal hypoxia as a result of cellular metabolism and counter current diffusion of oxygen between mucosal capillaries.5 In active UC, mucosal ischaemia and conversion of oxygen to reactive oxygen metabolites6 may further reduce colonic mucosal Po 2. Under such conditions, it seems likely that NOS independent pathways contribute to NO generation.

Figure 1

Production of nitric oxide (NO) from NO synthase (NOS) is oxygen dependent, but from xanthine oxidase (XO) occurs in hypoxic conditions. Reagents and buffers were pre-equilibrated with nitrogen. NOS (100 μM) was incubated with 1 mM l-arginine in buffer containing 50 mM HEPES (pH 7.4), 1 mM NADPH, 1 mM EDTA, 1.25 mM CaCl2, 1 mM dithiothreitol, and 10 μg/ml calmodulin (1). XO (70 μM) was incubated with 1 mM NADH and 1 mM nitrite (2). As controls, XO was incubated with nitrite (3), NADH with nitrite (4), and XO with NADH (5). Reactions were carried out in a 60 ml sealed flask with a reaction mixture volume of 500 μl. After incubation at 37°C for 20 hours, head-space gas was aspirated and injected directly into an NO analyser (TEA-502, Thermo Electron Corp, USA). Aerobic experiments were performed in a 25 ml flask linked to a NO-free air source, and hypoxic experiments were performed in an anaerobic cabinet (MDH, Hampshire) with aqueous O2 concentration < 1%. The mean of three experiments is shown.

In vivo rectal dialysis has shown excessive luminal accumulation of nitrite in active UC.7

Using homogenates of colonoscopic biopsy specimens from 10 patients with UC, we have found that, when incubated anaerobically with nitrite and NADH, inflamed mucosa produces NO (median NO generation 0.01, range 0.0047–0.033 μM/mg protein/min), detected directly with a chemiluminescence NO analyser.

Although it is possible that, in vivo, luminal bacterial nitrate/nitrite reductases catalyse this reaction, another candidate is the structurally similar mucosal enzyme, xanthine oxidase.8 This ubiquitous metalloflavoprotein is abundant in normal liver and small bowel, and catalyses the oxidation of NADH under hypoxic conditions to form NO with concomitant reduction of nitrite (fig 1). The activity of xanthine oxidase is increased in ischaemia–reperfusion injury and by activated neutrophils and a variety of inflammatory mediators present in inflamed gut mucosa. Using non-dissociated polyacrylamide gel electrophoresis with NADH activity staining, we have confirmed the presence of xanthine oxidase in homogenates of colonic biopsy samples from patients with UC.

As the mucosal production of hydrogen peroxide and reactive oxygen metabolites is notably increased in the inflamed bowel,6the recently described non-enzymatic pathway involving the reaction of hydrogen peroxide with both l- andd-arginine9 may be a third route by which NO is produced in UC.

Conventionally, NO generation in UC has been attributed predominantly to the activity of NO synthase.1-3 We suggest that, in the inflamed and hypoxic large bowel wall, important alternative sources include the reduction of nitrite by xanthine oxidase and bacterial nitrate/nitrite reductases, and the non-enzymatic reaction of hydrogen peroxide with arginine.


View Abstract

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.