Studies of dietary nitrate induced nitrosative stress in the upper gastrointestinal tract

Paterson, Stuart (2008) Studies of dietary nitrate induced nitrosative stress in the upper gastrointestinal tract. PhD thesis, University of Glasgow.

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The incidence of neoplasia at the gastroesophageal junction has increased markedly over the past 20 years, but the mutagen responsible for this remains unknown. Human saliva contains substantial quantities of nitrite and is the main source of nitrite entering the stomach. It is derived from the enterosalivary recirculation of dietary nitrate and its reduction by buccal bacteria. Carcinogenic N-nitrosocompounds may be formed from the nitrite in swallowed saliva by bacterially-catalysed nitrosation in the achlorhydric stomach or acid-catalysed nitrosation in the healthy acid secreting stomach. Protection against luminal acid-catalysed nitrosation is provided by the ascorbic acid content of human gastric juice which reduces nitrite to nitric oxide (NO). A high luminal concentration of NO generated from salivary nitrite has been shown at the gastroesophageal junction.

A bench top model has been developed to explore the chemistry occurring in an aqueous phase, representing the lumen at the gastroesophageal junction, after the addition of nitrite. Within this model an adjacent lipid phase has been created. The lipid phase represents the adjacent mucosa to the lumen or the dietary fat shown to sit within the fundus of the stomach. In this two-phase model, addition of physiological concentrations of nitrite to an acidic aqueous phase in the absence of ascorbic acid generated nitrosative stress within the aqueous phase. The addition of physiological concentrations of ascorbic acid to the aqueous phase prevents nitrosative stress within the aqueous phase but in so doing generates nitrosative stress within the lipid phase. This can be explained by the ascorbic acid converting the salivary nitrite to NO which diffuses into the lipid phase and there reacts with O2 to form the nitrosating species N2O3.
The bench top model has been developed further to include the lipid antioxidants, α-tocopherol, β-carotene and BHT. All three of these antioxidants are effective in inhibiting nitrosative stress within the lipid phase. The concentration of α-tocopherol required to inhibit nitrosation is less than would be expected from the reaction equation. A possible explanation for this is that ascorbic acid recycles the active antioxidant α-tocopherol from its reduced form. Further studies to support recycling of α-tocopherol by ascorbic acid are presented within this thesis.

The design of a model used in vivo to assess for nitrosative stress is discussed. The model consisted of a hydrophobic silastic tubing containing a secondary amine at a neutral pH. The hydrophobic membrane that the tubing is composed of allowed the passage of gaseous NO. Human studies in healthy volunteers were performed using the silastic tubing. The data presented shows that after a nitrate rich drink there is significantly more nitrosative stress within the upper gastrointestinal tract and this is particularly the case in the first part of the stomach where salivary nitrite meets acidic gastric juice containing ascorbic acid.

Finally, the studies explore directly measuring nitrosative stress in biopsy samples from humans and rats. As nitrite has been shown previously to be a marker of nitrosative stress in bench top models biopsy samples were assessed for nitrite and nitrate concentrations. Methods for assessing nitrite and nitrate concentrations within biopsy samples from the upper gastrointestinal tract are discussed, as nitrite and nitrate have not been analysed from this region previously. Thereafter, upper gastrointestinal biopsy samples from healthy human subjects were analysed after a control drink and a drink rich in nitrate. The studies presented show that biopsy nitrate and nitrite was higher in samples taken from the proximal stomach as compared to the oesophagus and distal stomach. This suggests that there is more nitrosative stress in the biopsies from the same area as where NO is generated after a nitrate meal.

Together the data presented supports a novel mechanism for N-nitrosation in the upper gastrointestinal tract of a healthy subject. The N-nitrosation is maximal where swallowed salivary nitrite from dietary nitrate meets acidic gastric juice containing the aqueous antioxidant ascorbic acid.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Nitrosamines, Oesophagogastric cancer, Nitric Oxide, Ascorbic acid, Tocopherol, Nitrosative Stress, Dietary Nitrate, Nitrite, Barrett's oesophagus, Lipid
Subjects: R Medicine > R Medicine (General)
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Q Science > QD Chemistry
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
Supervisor's Name: McColl, Professor Kenneth E.L.
Date of Award: 2008
Depositing User: Dr Stuart Paterson
Unique ID: glathesis:2008-394
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 05 Jun 2009
Last Modified: 10 Dec 2012 13:18

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