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Listen to this siteWednesday 24 March 2004
This research project proposes to determine the chemical nature of faecal ATNC together with its route of formation.
Study Duration : October 2002 to June 2004
Contractor : Central Science Laboratory
A diet high in red meat has been shown to be associated with increased levels of colorectal cancer. It has also been shown to be associated with the increased levels of non-volatile N-nitroso compounds (NOC) formed by nitrosation in the gut. These are measured as apparent total nitroso-compounds (ATNC) in faecal extracts. Increased intakes of red meat have been shown to increase faecal ATNC levels in a dose responsive manner, the equivalent amount of protein from eggs, milk, cheese and vegetable protein have been shown to have no effect. Although many NOC are known carcinogens, the content of ATNC is poorly defined and its biological relevance unknown.
Faecal ATNC will be separated into water soluble, dichloromethane soluble and insoluble fractions. The dichloromethane fraction will be further identified by gas chromatography-mass spectrometry (GC-MS). The water soluble fraction will be characterised by molecular weight profile using aqueous size exclusion chromatography. Lower molecular weight fractions will be characterised by liquid chromatography-mass spectrometry (LC-MS). Higher molecular fragments will be enzymatically digested and digest characterised by LC-MS. N-Nitrosoproline and a synthesised N-nitrosopeptide will be used for standardisation.
This project is a collaboration with project T01030 - 'Biological relevance of apparent total nitroso compounds in the human colon'.
This project (a collaboration with T01030*) set out to isolate and identify one or more individual NOC in ATNC isolated from faecal extracts. Subsequently, analysis was extended to include ileostomy fluid which has been shown to have a higher concentration of ATNC and a range of components with different mass spectra to those from faecal extracts.
Nitrosation in the digestive tract is probably non-specific i.e. level of product depends on the amount of parent amine. Therefore faecal water and ileostomy fluid samples were nitrosated prior to analysis to increase the concentration of ATNC and thus the chances of isolating individual NOC without altering their relative ratios. This nitrosation led to a considerable increase in the ATNC content of both sample types. Prior to nitrosation, faecal water samples from subjects consuming a high haem (red) meat diet had higher ATNC content than samples from subjects consuming a vegetarian diet. After nitrosation, these samples had similar levels of ATNC as expected as levels of ATNC precursor in both diets are similar.
Nitrosation was confirmed by comparing un-nitrosated samples with samples nitrosated using either 14N or 15N nitrite. Peaks corresponding to nitrosation products could then be identified and MS/MS analysis would show two peaks with one 1 mass unit difference between the 14N and 15N spectra, confirming nitrosation. UV light was used to confirm N-nitrosation as these compounds will degrade following irradiation whereas C-nitroso-compounds do not.
Chromatograms of faecal water samples had fewer peaks with evidence of nitrosation and the MS spectra were weaker in intensity, compared to those from ileostomy fluid samples. This indicated that either the faecal samples were so complex that they could not be analysed effectively using this method of LC-MS, or there is degradation of nitrosamines in the digestive tract after the ileum.
This project presented a very challenging task which did not meet all its objectives. Unfortunately it was not possible to identify specific individual N-nitroso compounds or generate a range of standards to complement the one compound available commercially (nitrosoproline). Ileostomy fluid samples showed evidence of a number of different nitroso-compounds. An N-nitrosocompound was detected consistently in a sample of faecal water analysed on more than one occasion. This was confirmed as an N- and not a C-nitroso-compound on the basis that the peak disappeared when UV irradiated. However, due to technical problems further analysis of this sample was not possible.
*Project completed - Final report is awaited.
*Project completed - Final report is currently being evaluated by the Agency.
*The final report is available from the Agency’s Information Centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email:
infocentre@foodstandards.gsi.gov.uk
)
For any enquiries concerning this research project, please contact the relevant Programme contact or email: science@foodstandards.gsi.gov.uk
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