Direct measurement of nitric oxide (NO) in the gastrointestinal tract of cod (Gadus morhua)

Abstract Objective: In mammals, the biological messenger nitric oxide (NO) is generated throughout the gastrointestinal (GI) tract from the reduction of dietary nitrate and nitrite. The aim of the present study was to investigate the amount of GI NO in Atlantic cod (Gadus morhua) in relation to intake of food. Methods: A total of 28 cod were divided into 3 groups, fed at different times before the experiment (1 week, 1 day, and 3 h, respectively). Results: In the stomach, the measured NO concentrations were consistently higher in the group fed 3 h before the measurement, implying that the NO3-NO2-NO pathway is present in the stomach of cod. We also measured the NO concentration in the large intestine. Again, the values were higher in cod fed 3 h before the experiment. Conclusion: We conclude that NO is formed in the GI tract of cod, likely via the reduction of dietary nitrate and nitrite. The physiological importance of this NO production remains to be determined.


Introduction
Over the years, nitrate (NO 3 ) and nitrite (NO 2 ) have been regarded as undesired residues in the food chain with potentially carcinogenic effects or as inert oxidative end products of endogenous nitric oxide (NO) metabolism, and great efforts have been made to reduce the amounts of these compounds in food and water supplies. In fi sh, nitrite in water is believed to be absorbed through the gills following the same pathways as sodium chloride, possibly leading to chloride depletion (1,2). Furthermore, nitrite is also believed to interact with hemoglobin (2,3). Both mechanisms might be detrimental to fi sh health.
However, recent comparative studies in germfree and conventional mammals (4,5) have shown that nitrate and nitrite are both part of a physiological nitrogen recycling system in which salivary glands and the microbial fl ora in the digestive tract interact to reduce nitrate and nitrite to NO. Dietary nitrate is rapidly absorbed and excreted into saliva within a few minutes. Bacterial conversion of nitrate to nitrite starts in the mouth and a further, pH-dependent, conversion of nitrite to NO takes place in the stomach (5). It has also been shown that the amount of NO in the digestive tract was related to dietary intake of nitrate (5). The possible physiological impact of this microbial-derived NO production in the digestive tract has recently been reviewed (6).
To the best of our knowledge, the presence of NO in the digestive tract of fi sh has never been studied. The aim of the present study was to investigate whether NO is present in the stomach of Atlantic cod (Gadus morhua), and whether the amount is infl uenced by the feeding regimen. We also measured the NO concentration in the large intestine.

Material and methods
The investigation took place under fi eld conditions at a cod farm, located north of the Polar Circle in the county of Nordland, Norway. Water temperature in the ponds was about 9°C. The conditions at the location limited the number and types of samples that could be taken for investigation.
cod. The consistent differences in NO concentrations found in the three groups of cod investigated implies that also in cod, NO production in the GI tract is related to food intake. The very low values found in the LT group imply that a majority of the food had already been digested, whereas the higher values found in the MT and ST groups indicated that digestion was still going on.
The large intestine generally contained more NO in the MT than in the ST group, indicating that the food had not been properly digested yet in the ST group. This is also consistent with observations made at the site of the experiments: the variations in the amount of stomach and large intestine were The fi sh investigated were raised in captivity and fed a commercially obtained fi sh diet, and they were of the same size, approximate weight 3 kg. The groups studied were in three different ponds. Each cod was taken individually from the pond before investigation, thereby standardizing the time that they were out of water. The three groups were as follows. Group 1, avoided food for a week (LT ϭ long time); group 2, avoided food for 20-24 h (MT ϭ medium time); and group 3, avoided food for the last 2-4 h (ST ϭ short time).
After removal from the water, the cod was immediately killed by a blow to the head, the abdominal wall was opened, and the stomach was isolated by sealing the esophagus and duodenum, using external clamps. Thereafter, 4 ml of air was inserted by means of a 5 ml syringe and a thin needle into the stomach. After about 10 s, the gas was ejected from the stomach and immediately injected into a rapidresponse chemiluminescence analyzer (Aerocrines AB, Stockholm, Sweden) to determine the concentration. The instrument's detection level for NO was 1 part per billion (ppb), and the response time was Ͻ 0.5 s. Measurement of NO took place Ͻ 60 s after the cod was killed.
In some fi shes, part of the large intestine was also isolated as described for the stomach, and 3 ml of air was inserted. After 10 s, gas was ejected and the content of NO was measured as described above.
Except for individual and rapid handling, the procedures for harvesting the cod and opening of abdominal wall followed the ethical rules at the farm.

Results
As can be seen by the data presented in Figure 1, very low levels of NO were found in the LT group (medium value 1.4 ppb), whereas signifi cantly higher values with considerable variations were found in the ST group; the values differed in the MT and ST groups (median values 8 and 65 ppb, respectively).
A few measurements of NO were also made in the large intestine (Figure 2), and here the highest values were found in the MT group.
The nitrate content measured in the food peaked at 32 μM or 2 mg/kg.

Discussion
As mentioned above, it is now well established in mammals that the nitrate-nitrite-nitric oxide pathway is responsible for a substantial part of NO present in the lumen of the GI tract (6). Our fi ndings indicate that similar mechanisms are also at work in Figure 1. Logarithmic values of NO concentration in stomachs of cod measured in ppb. The fi sh were divided into three groups: group 1 was on starvation, group 2 had been fed 24 h before the experiment, and group 3 was fed 3 h before the experiment. Mean values are indicated. Figure 2. Logarithmic values of NO concentration in the large intestine of cod measured in ppb. The fi sh were divided into three groups: group 1 was on starvation, group 2 had been fed 24 h before the experiment, and group 3 was fed 3 h before the experiment. Mean values are indicated.
is likely the major substrate, and NO generation probably occurs via serial reduction to nitrite and then NO. It remains to be studied whether NO plays an equally important role in regulation of GI function in fi sh as it does in mammals. most pronounced in the ST group. Variations in amount of content -as well as the amount of NO in the stomach and the large intestine -are probably related to some eating habits in farmed cod: they are eating in a hierarchic order and they may retain food in the mouth for hours before swallowing. The farmers try to counteract these habits by expanding the feeding period. Although these habits are issues of importance to the farmers, they will not be stressed further in this article. Instead, we simply note that future study would benefi t from a more controlled feeding procedure, where the exact feeding time for each individual can be monitored.
The nitrate concentration found in the diet was low compared with the concentration found in most mammalian food. For example, the nitrate concentrations in vegetables such as spinach, lettuce, and beetroot are all approximately in the range of 2000-3000 mg/kg, and the estimated mean intake of dietary nitrate in European adults is 31-185 mg/day (6). In future experiments, it would be of interest to vary the exact amount of nitrate fed to each individual cod in order to quantitatively measure the rate at which nitrate is reduced to NO.
It should be mentioned that the nitrate-nitritenitric oxide cycle is not the only pathway for production of nitric oxide in fi sh. It has long been known that arginine-dependent NO synthases (NOSs) are present endogenously in fi sh. In a previous study, we reported upon the presence of NO in the swim bladder of Atlantic cod (7). As the cod is a physoclist, i.e. has a closed, sterile swim bladder, we could exclude the involvement of microbes in NO production in that compartment.
We conclude that NO can be formed in the stomach and large intestine of Atlantic cod. Dietary nitrate