Fat transforms ascorbic acid from inhibiting to promoting acid‐catalysed N‐nitrosation
E Combet, S Paterson, K Iijima, J Winter, W Mullen, A Crozier, T Preston, and K E L McColl
Gut. 2007 December; 56(12): 1678–1684.
Background
Over the last 20 years, a new pattern of gastric carcinogenesis has emerged in the western world, with a decreasing incidence of distal gastric cancer and an alarming increase in the incidence of adenocarcinomas of the proximal stomach, including cardia and adjacent gastro‐oesophageal junction (GOJ).1,2,3,4 The cancers at the GOJ usually occur in healthy acid‐secreting stomachs.5,6 The cause of the increasing incidence of adenocarcinoma of the proximal stomach remains unclear, but the rate of change indicates environmental factors.
For many years, there has been interest in the potential for endogenous generation of carcinogenic N‐nitroso compounds from nitrite within the human upper gastrointestinal tract. This is due to the fact that the acidic pH of gastric juice converts nitrite to nitrous acid and nitrosating species such as N2O3, NO+.7,8 The latter reacts with thiocyanate which is also present in gastric juice to form the particularly potent nitrosating species NOSCN.9,10,11 These nitrosating species can react with secondary amines and amides to form N‐nitroso compounds, many of which are carcinogenic and widely used in animal models of cancer.8,12,13,14,15,16 The nitrosating species N2O3 is itself mutagenic as it can directly deaminate certain DNA bases and inactivate important DNA repair enzymes.17,18,19,20,21 Understanding the factors affecting gastric nitrite chemistry is therefore relevant to our understanding the development of malignancies of the upper stomach.
The main source of nitrite entering the stomach is swallowed saliva. The high level of nitrite in saliva (100 µM under fasting conditions) is derived from the enterosalivary recirculation of dietary nitrate and its reduction to nitrite by buccal bacteria.22,23,24,25,26,27 Consequently, the nitrite level in saliva rises several fold for at least 2 h after ingesting nitrate‐containing foodstuffs. The nitrite concentration in the distal oesophagus is similar to that in saliva.28
A major factor protecting against the generation of N‐nitroso compounds from salivary nitrite on entering the acidic stomach is ascorbic acid present in gastric juice.29,30,31,32,33 Ascorbic acid is actively secreted in gastric juice and effectively competes with secondary amines and amides for reaction with the nitrosating species.32,34,35,36,37,38 In this reaction, the nitrosating species are reduced to nitric oxide and the ascorbic acid oxidised to dehydroascorbic acid.39 We and others have recently demonstrated that high concentrations of nitric oxide are generated in the human upper gastrointestinal tract following nitrate intake by the above mechanism.40
This reduction of acidified nitrite to nitric oxide has been regarded as an effective mechanism for protecting against the generation of N‐nitroso compounds. However, it has recently been recognised that nitric oxide can generate nitrosating species. This arises from the ability of nitric oxide to react with molecular oxygen to form N2O3 – the same species formed by acidification of nitrite.18,41,42 The rate of the reaction between nitric oxide and oxygen to produce N2O3 is proportional to the concentration of oxygen and to the square of nitric oxide concentration.43,44 Consequently, this reaction is most important at high nitric oxide concentrations.
Recent studies have also demonstrated that the reaction between nitric oxide and oxygen is 300 times faster within lipid than within an aqueous phase.41 This is due to the fact that nitric oxide is nine times more soluble in lipid, and oxygen is also more soluble in lipid than aqueous solutions, resulting in both reactants accumulating in the lipid compartment.41 The potential for generation of nitrosating species from nitric oxide will therefore be greatest when high concentrations of nitric oxide are generated close to lipids.
The above chemistry raises the possibility that inhibition of nitrosation reactions by ascorbic acid in the aqueous phase may promote nitrosation within adjacent lipid compartments. This could occur by diffusion of the nitric oxide produced by the reaction between acidified nitrite and ascorbic acid, into adjacent lipid compartments and their reacting with oxygen to form the nitrosating species N2O3. The presence of lipid might therefore have a profound effect on the chemistry occurring between acidified nitrite and ascorbic acid.
We have demonstrated that luminal nitrosative chemistry in the acid‐secreting stomach is maximal where swallowed saliva first encounters acidic gastric juice and may therefore be contributing to the high incidence of metaplasia and neoplasia in the proximal stomach and GOJ.28,40 The aim of this study was to investigate the influence of lipids on this luminal nitrosative chemistry.
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Discussion
These studies indicate that the presence of lipid profoundly alters acid‐catalysed nitrosative chemistry. The lipid phase is able to convert the influence of ascorbic acid from one that protects against nitrosation to one that promotes it. This effect is likely to be due to the ability of the nitric oxide formed by ascorbic acid within the aqueous phase to regenerate nitrosative species by reacting with oxygen within the lipid phase.
In the studies performed without lipid, and without ascorbic acid, the addition of nitrite to the HCl pH 1.5 containing thiocyanate resulted in nitrosation of the secondary amines. The main nitrosating species formed under these conditions is NOSCN.9,10,11 NOSCN reacts with the secondary amine in its unprotonated uncharged state.48 The amount of the secondary amine in its nitrosatable form depends upon its −log dissociation constant (pKa). The pKa values of the amines studied are 8.33 for morpholine, 11.22 for piperidine, 10.73 for dimethyamine and 11.09 for diethylamine. The amount of the secondary amine in a form available for nitrosation by NOSCN will therefore be greatest for morpholine and least for piperidine. The differences in pKa can partially explain why different concentrations of the four N‐nitrosamines were generated.
In the experiments performed in the absence of lipid, the addition of ascorbic acid effectively prevented the nitrosation of the amines. This can be explained by the ascorbic acid competing with the secondary amines for the NOSCN.36,38,49 In the reaction between ascorbic acid and NOSCN, the latter is reduced to nitric oxide and the former oxidised to dehydroascorbic acid. Consistent with this, we observed a burst of nitric oxide accompanied by a fall in the ascorbic acid and oxygen concentrations. This can be explained by the nitric oxide reacting with dissolved oxygen to form N2O3.43,44 The N2O3 formed in this way is a nitrosating species and again preferentially reacts with ascorbic acid and is reduced back to nitric oxide.39 This recycling continues until either the oxygen or the ascorbic acid is consumed. Under our experimental conditions, the oxygen was the first to be depleted. Stoichemically, 1 mol ascorbic acid can reduce 2 mol nitrite to nitric oxide. The greater consumption of ascorbic acid observed is due to this recycling of nitric oxide.
In the presence of the lipid, N‐nitrosamines were formed despite the presence of ascorbic acid. Indeed, the presence of lipid transformed the effect of the ascorbic acid from effectively inhibiting nitrosation of each amine to powerfully enhancing nitrosation of three of the four amines. (The overall amounts of NDMA, NDEA and NPIP in the system were increased by 8‐, 60‐ and 140‐fold, respectively, in the dual‐phase system when ascorbic acid was present versus absent; fig 33.).) The overall amount of NMOR formation was inhibited by ascorbic acid in both the absence and presence of lipid, although the inhibitory effect of ascorbic acid was markedly reduced by the presence of lipid, from over 1000‐fold in absence of lipid, to only threefold in the presence of lipids. In addition, the presence of ascorbic acid increased the concentration of NMOR in the lipid from undetectable to 4.9±0.4 µM.
What is the explanation for the ability of the lipid to convert the effect of the ascorbic acid from being an inhibitor to a promoter of nitrosation? The effect observed is likely to be mediated by the nitric oxide produced by the reaction between the ascorbic acid and the nitrosating species within the aqueous phase. The nitric oxide will diffuse into to the lipid phase and within it react with oxygen to form N2O3.50 Liu et al. demonstrated that the reaction between nitric oxide and oxygen is 300 times faster in lipid than in aqueous solutions, due to the increased solubility of both gases in lipids.41 As ascorbic acid is not lipophilic, it is unable to enter the lipid and thus the N2O3 generated within the lipid will be able to nitrosate the secondary amines within the lipid. The N‐nitrosamines generated within the lipid in this way will then diffuse out of the lipid and produce the rise in their concentration observed in the aqueous compartment (fig 55).
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In the dual‐phase studies without ascorbic acid, low concentrations of NDMA and NPIP were detected in the lipid phase, whereas NDEA and NMOR were both undetectable in the lipid phase. NDMA and NPIP are more lipid soluble than NMOR (ClogP 0.01, 0.73 and −0.33, respectively) and are likely to have diffused into the lipid following their generation in the aqueous phase. Low levels of nitric oxide are present in the aqueous phase even in the absence of ascorbic acid and this may also have nitrosated dimethylamine and piperidine within the lipid phase. The dual‐phase experiments with ascorbic acid indicated that dimethylamine and piperidine were the amines most nitrosated by nitric oxide in the lipid phase. Piperidine showed the greatest degree of nitrosation in the dual phase; this may be due to its lipophilicity (ClogP 0.52) and thus it is the one most available for nitrosation within the lipid compartment.51
In the dual‐phase system, the concentrations of three of the N‐nitrosamines detected in the aqueous phase were significantly greater in the presence versus absence of ascorbic acid. The one exception was NMOR and a number of factors may explain this. First, the amount of NMOR formed in the aqueous phase in the absence of ascorbic acid is considerably greater than the amount of other N‐nitrosamines, due to its lower pKa and thus higher proportion in the nitrosatable unprotonated form.52 Second, the aqueous concentrations of each N‐nitrosamine were similar in presence of both ascorbic acid and lipid compared to aqueous concentrations in the absence of ascorbic acid (table 22).). This is because the N‐nitrosamines detected in the aqueous phase in presence of both ascorbic acid and lipid will have been formed mainly in the lipid phase, where the pKa of the amine is not relevant. The decrease of NMOR aqueous concentration in the dual‐phase system in presence of ascorbic acid compared to when ascorbic acid is absent is therefore mainly explained by the inhibition of the aqueous nitrosation of morpholine when ascorbic acid is present. In addition, differences in partitioning of the different amines and N‐nitrosamines between the lipid and aqueous phase may contribute to the different results.
The above studies indicate that the presence of lipid transforms the regulation of nitrosative chemistry in conditions simulating the proximal stomach. The presence of lipid overcomes the protective effect of ascorbic acid and indeed transforms ascorbic acid from an inhibitor to a promoter of nitrosation. The transforming role of lipids is likely to be relevant to the in vivo situation as lipid is present in the proximal stomach for a considerable time after eating and is also an important component of the epithelial membranes.
