Growth of lettuce with different content of inorganic nitrate as a feeding strategy for placebo-controlled nutritional interventions to test the effects of inorganic nitrate on human health

Abstract

The chemical composition of vegetables is dependent on several growing conditions. This effect relates to phytochemical compounds including secondary metabolites and other bioactive non-nutritional compounds. This study aimed to use different nitrogen fertilizer regimes to produce vegetables with so large differences in nitrate content that they can be used as treatment and placebo to study the effect of nitrate on human health. Green leafy vegetables, such as lettuce or rocket, are rich in inorganic nitrate (NO3) and an increased consumption has been associated with beneficial effects on blood pressure (BP). The objective of this study was to compare whether two lettuce materials with controlled high and low NO3 content may have different effects on BP in healthy subjects. Firstly a gas chromatography mass spectrometry (GC-MS) method using pentafluorobenzyl bromide and chemical ionisation to determine nitrate and nitrite levels was modified to optimise the analytical process. This method has been used to measure samples of lettuce as well as samples of human plasma, urine and saliva obtained from this study and several other projects. In this study, two sets of lettuce were produced as a model vegetable with high and low nitrogen fertiliser in controlled growing conditions to manipulate nitrate concentration of the vegetable as a tool for investigating the effect of nitrate content of food on human health. This was tested by determining the correlations between nitrate intake and blood pressure (BP), by measuring the short effect on systolic and diastolic BP and nitrate and nitrite contents in blood, urine and saliva samples. The novelty of this study is that the placebo and the treatment (lettuce with low and high nitrate content, respectively) have similar appearance despite being very different in nitrate content, making it possible to blind the subjects to the treatment and control placebo effects of vegetable consumption. Using 26 and 154ppm ammonium nitrate in the fertigation solution, lettuce was produced with a high (~530 mg nitrate/50g FW portion) or low (~3 mg/50g) nitrate content. However not all confounding factors could be controlled, e.g. the low nitrate lettuce produced a high amount of phenolic acids which was 69.5 mg/50g FW and high nitrate lettuce produced a much lower amount which10.5mg/50g FW (P<0.001). So while the nitrate content can be manipulated substantially via fertilizer treatments, the low-nitrate lettuce also differs from the high-nitrate lettuce in the content of other non-nutrient phytochemicals. An intervention trial was carried out with twenty healthy young volunteers (12 females and 8 males) in a randomised, double-blind placebo controlled cross-over design, with two 24-hour ii intervention phases separated by a 3-week washout period to avoid carry-over effects. Blood pressure was recorded by 24-hour ambulatory blood pressure monitoring (ABPM), every 30 mins during day-time and every 60 min at night-time. Consumption of high nitrate lettuce significantly increased plasma NO3 (3 hrs post-ingestion: 395±133 μM; P<0.001), salivary NO3 (3 hrs post-ingestion: 7362±4666 μM; P<0.001) and urinary NO3 (3 hrs post-ingestion: 2287±1233μM; P<0.001) concentrations whereas non-significant changes were measured in the low nitrate lettuce group. High nitrate lettuce significantly reduced systolic BP (-2.80±4.43 mmHg and -6.85±4.91 mmHg after 3 and 6 hrs, respectively; P=0.003) and diastolic BP (-2.25±2.34mmHg and Δ3.85±3.01 mmHg after 3h and 6 hrs, respectively; P=0.002) compared to low nitrate lettuce. No significant difference was observed between high nitrate lettuce and low nitrate lettuce for TEAC (P = 0.32), FRAP (P = 0.26), cGMP (P = 0.19) and plasma concentrations of phenolic compounds. The results showed that the amount of urinary nitrate excreted during a 24-hour period increased from 104mg with low nitrate lettuce to 391mg (P<0.001) with the high nitrate lettuce. In conclusion, ingestion of high nitrate lettuce significantly increases plasma, saliva and urinary nitrate and lowers systolic and diastolic BP compared to low nitrate lettuce. The development of NO3-enriched and NO3-depleted vegetable products with similar organoleptic characteristics could provide a unique opportunity to conduct double-blind nutritional interventions and advance knowledge on the role of dietary nitrate on human health.