C01013: Dietary nitrate consumption: an analysis of biomarkers of DNA and protein damage in humans

Study Duration: September 1999 to January 2003

Contractor: King's College London / University College London

Certain foods, such as green leafy vegetables, contain a lot of inorganic nitrate. For a long time, there has been a theoretical concern that nitrate in foods (or water) may react with gastric acid or other components of the diet to create reactive nitrogen species (RNS). Some RNS are known to cause cancer, but others may be beneficial. Although the possible harmful effects of RNS (including stomach cancer) have received extensive publicity, epidemiological studies (comparing groups of people who have been heavily exposed to nitrate in the diet with those who have not) do not show any measurable risk. Therefore, the Food Standards Agency has been actively looking for evidence to support or refute the idea that nitrate in the diet can be harmful to man.

Recent studies have shown that nitrate can be converted, by bacteria normally present in the back of the mouth, into nitrite, which then reacts with acid in the stomach to form RNS. These may be good, in that some RNS are known to kill infective organisms, and to have an 'aspirin' like effect by inhibiting the formation of blood clots. This may be one reason why people who eat a balanced diet, which usually contains a lot of green leafy vegetables, and is therefore rich in nitrate, are found to have a decreased risk of coronary heart disease. However, there is also the potential for other RNS to cause damage to DNA and proteins. Thus it is essential to understand the balance between the potential benefits and toxicity of dietary nitrate, in relation to the current recommended UK limits for dietary intake.

The three major aims were:

• To determine the effect that dietary nitrate from a whole round green lettuce (British produce) had on the levels of nitrate and nitrite in blood, saliva, gastric juice and urine in normal healthy volunteers. The consumption of a whole lettuce at a single sitting gives a nitrate intake at the upper limit of current recommendations for daily consumption (225mg).
• To determine whether a high nitrate intake causes protein nitration or DNA damage in normal volunteers, and whether it has an 'aspirin' like effect and inhibits platelet clotting ability in healthy volunteers.
• To investigate whether gastric acidity or stomach infection influence nitrate metabolism compared with normal healthy volunteers. This is important since Helicobacter pylori infection (which may cause stomach ulcers) is present in between 30-50% of the adult population, and many people take drugs that decrease the acidity of their stomach.

Approach

For the first two aims 30 normal healthy volunteers, aged 20-50 years, were recruited. In the 24 hours before the study, volunteers were maintained on low nitrate diet. The volunteers then ate about 4-5 ounces (120g) of green lettuce (equivalent to 225 mg nitrate) at lunch time, along with other foods that contained very little nitrate, for 3 consecutive days. Evening meals were nitrate free. Thus patients were exposed to a large 'dose' of nitrate at lunch time, representing an extreme dietary intake (not many people eat a whole lettuce for lunch).

During this time, all urine was collected, and blood and saliva samples were taken at specific times before and after the nitrate meals. Blood, saliva, and urine were analysed for nitrate and nitrite levels each time. Biomarkers for protein damage (formation of nitrotyrosine) or DNA damage (modified bases) were measured in the blood samples. The effect on an index of blood clotting was also determined as a marker for the possible effects on blood.

To cover the third aim, 18 normal healthy subjects and 12 patients with Helicobacter pylori infection were studied. After a low nitrate diet day, all volunteers and patients ate the same high nitrate meal as above (120g lettuce) on two consecutive days. However, after the first high-nitrate day, subjects took two tablets of omeprazole, a drug commonly used in the UK to treat ulcers and inhibit gastric acid secretion. Thus, they had a lower amount of gastric acid during the second day when they ate the high nitrate meal. A decrease in gastric acid secretion might be expected to prevent the formation of reactive nitrogen species. It was important to study normal subjects on omeprazole, as well as patients, not only to determine whether the groups reacted differently but also to establish whether reduced acidity (which is often self-induced with antacid tablets as well as by prescribed treatment) influenced the outcome for the normal subjects.

Gastric juice, blood, urine and saliva samples were collected at regular intervals and analysed for the presence of biomarkers of RNS formation. We developed a new method to assess the formation of RNS in vivo, which relied on the observation that a compound present in saliva (4-hydroxypenylacetic acid (PHPA)) is readily nitrated in the test tube.

For a long time, there has been a theoretical concern that nitrate in foods (or water) may react with gastric acid or other components of the diet to create reactive nitrogen species (RNS). RNS can potentially cause damage to DNA and proteins; hence it is essential to understand the balance between the potential benefits and toxicity of dietary nitrate, in relation to the current recommended UK limits for dietary intake. One of the ways RNS damage proteins is by reacting with tyrosine (an amino acid that constitutes about 5% of all proteins) in the protein to form nitrotyrosine. This may alter the protein function or stop it working altogether. The nitrotyrosine can be recovered after the protein is broken down by natural processes. The amount present can thus act as a 'biomarker' for protein damage. Similarly, altered bases from DNA can be used as biomarkers for DNA damage. This study has used these biomarkers and direct measurements of nitrate and nitrite to assess the balance of effects following a meal high in nitrate.

The three major aims were to determine the effect that dietary nitrate from a whole round green lettuce had on the levels of nitrate and nitrite in blood, saliva, gastric juice and urine in normal healthy volunteers; to determine whether a high nitrate intake causes protein nitration or DNA damage in normal volunteers, and whether it has an aspirin-like effect and inhibits platelet clotting ability in healthy volunteers; and to investigate whether gastric acidity or stomach infection influence nitrate metabolism compared with normal healthy volunteers. This is important since Helicobacter pylori infection (which may cause stomach ulcers) is present in between 30-50% of the adult population, and many people take drugs that decrease the acidity of their stomach.

As expected, it was demonstrated that a high nitrate meal leads to a rapid increase in the blood, urinary and salivary nitrate concentrations, with a roughly four-fold increase in urinary nitrate excretion, and a seven-fold increase in plasma nitrate concentrations at 1-4 hours after eating the high nitrate meal. Urinary nitrate excretion returned to normal by 24 hours, and the majority of the ingested dietary nitrate was excreted and recovered in the urine within 24 hours. There were no corresponding changes in the levels of biomarkers of protein nitration or DNA damage, or blood clotting ability observed during this study. This established that there is no significant protein nitration or DNA damage after a high nitrate meal.

However, one possibility that concerned the researchers was that the methods employed to look for protein nitration were not sensitive enough. Previous studies have shown that following release from protein, nitrotyrosine may be metabolised into 3-nitro, 4-hydroxyphenylacetic acid (NHPA). This compound can also be formed by nitration of 4-hydroxyphenylacetic acid (PHPA), which is present in saliva. Thus, measurement of urinary NHPA should be a good marker for the formation of RNS in vivo. A method was developed using mass spectrometry to measure the levels of NHPA in the urine as a biomarker of RNS.

These studies demonstrate that the normal recommended dietary levels of nitrate do not produce measurable amounts of the biomarkers selected. This implies that these levels are safe and non-toxic in the normal population. Further examination of the effect of nitrate in H. pylori patients (who may be up to 40% of the population) is required, although these preliminary studies suggest an enhanced benefit. Since plasma nitrate increases to a considerably greater extent in these patients compared with normal subjects on dietary nitrate administration, it is possible that these levels could offer some protection against platelet dysfunction, as seen with a single bolus supplement of inorganic nitrate in other reported studies.

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)

Contact: For any enquiries concerning this research project, please contact the relevant programme contact or email: science@foodstandards.gsi.gov.uk