Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite
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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These authors contributed equally.