Elsevier

Nitric Oxide

Volume 13, Issue 4, December 2005, Pages 272-278
Nitric Oxide

Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite

https://doi.org/10.1016/j.niox.2005.08.002Get rights and content

Abstract

Denitrifying bacteria in soil generate nitric oxide (NO) from nitrite as a part of the nitrogen cycle, but little is known about NO production by commensal bacteria. We used a chemiluminescence assay to explore if human faeces and different representative gut bacteria are able to generate NO. Bacteria were incubated anaerobically in gas-tight bags, with or without nitrate or nitrite in the growth medium. In addition, luminal NO levels were measured in vivo in the intestines in germ-free and conventional rats, and in rats mono-associated with lactobacilli. We show that human faeces can generate NO after nitrate or nitrite supplementation. Lactobacilli and bifidobacteria generated much NO from nitrite, but only a few of the tested strains produced NO from nitrate and at much lower levels. In contrast, Escherichia coli, Bacteroides thetaiotaomicron, and Clostridium difficile did not produce significant amounts of NO either with nitrate or nitrite. NO generation in the gut lumen was also observed in vivo in conventional rats but not in germ-free rats or in rats mono-associated with lactobacilli. We conclude that NO can be generated by the anaerobic gut flora in the presence of nitrate or nitrite. Future studies will reveal its biological significance in regulation of gastrointestinal integrity.

Section snippets

Faecal samples

Faeces was obtained from healthy volunteers (n = 8 age 28–46 years, 5 females and 3 males). One gram wet weight was suspended in 9 ml sterile 0.9% NaCl and 10-fold serial dilutions were made. From the 10−2 dilution, 100 μl per plate was inoculated on three different ISO-sensitest agar plates (Oxoid, Basingstoke, England): regular ISO-sensitest agar or ISO-sensitest agar supplemented with 0.1 mM NaNO2 or 0.1 mM NaNO3. After bacterial inoculation, the plates were inserted into 500 ml gas tight infusion

NO generation from the faecal flora

The faecal flora of healthy volunteers generated small amounts (∼75 ppb) of NO after 24 h of anaerobic incubation without supplementation of nitrite or nitrate to the medium. This NO production was greatly increased when nitrite (∼2000 ppb) or nitrate (∼900 ppb) was added to the medium. Kinetic experiments showed that the NO production from faecal flora reached a plateau at 24 h, therefore this time point was chosen for the subsequent measurements (Fig. 1). There was a large individual variation in

Discussion

Bacterial generation of NO can occur via several pathways. First, NO can be generated as a part of the denitrification pathway via nitrite reductases [7]. Second, NO can form non-enzymatically from nitrite via acidic reduction [16], [17], [18]. Third, there have been reports of NO generation from l-arginine via bacterial NO synthases. We believe that the first two pathways are the predominant sources of NO and that in some situations they may operate together. In this study, we found a clear

Acknowledgments

This work was supported by grant from The Knowledge Foundation, The Swedish Research Council, The Ekhaga foundation, the EU 6th framework program and the Centre for Allergy Research, Karolinska Institutet, Bengt E. Gustafsson fond 2000. The authors would like to thank Prof. J. Cole, Birmingham, UK for his invaluable help in reading this manuscript, and Dr. D. Jonkers, Dr. E. Stobberingh and Prof. R. Stockbrügger (Departments of Medical Microbiology and Gastroenterology, University Hospital

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