Consumption of Nitrate-Rich Beetroot Juice with or without Vitamin C Supplementation Increases the Excretion of Urinary Nitrate, Nitrite, and N-nitroso Compounds in Humans

Abstract

Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, following a secondary pathway, NO may also lead to the formation of N-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total N-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol: p < 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol: p < 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol, p < 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete's health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.

Keywords: Beetroot juice; N-nitroso compounds; human dietary intervention; nitrate; nitrite; vitamin C.

Figure 1

Metabolic pathway of nitrate (NO₃⁻), nitrite (NO₂⁻), nitric oxide (NO), nitrosamines, nitrosamides, and the effect of vitamin C. Nitric oxide (NO) is mainly responsible for the physiological effects of beetroot juice (BRJ). The body uses arginine (C₆H₁₄N₄O₂) as a source to form NO, and for this reaction oxygen is needed. However, NO can also be formed after intake of nitrate-rich products such as BRJ. Ingested nitrate (NO₃⁻) will be partly reduced to nitrite (NO₂⁻) by microflora in the oral cavity. In oxygen-poor environments nitrate and nitrite can be reduced into NO. NO can also be oxidized back into nitrate and nitrite which are water soluble and can therefore be excreted in urine. Under acidic conditions, such as in the human stomach, nitrite will react with the H⁺ and will form HNO₂ (nitrous acid). Also in the stomach, two molecules HNO₂ can form N₂O₃ (dinitrogen trioxide), by proton catalysis. N₂O₃ plays a role in the N-nitrosation rate. Increasing the amount of nitrate will therefore lead to an increase in the N-nitrosation rate. Subsequently, HNO₂ can react with amides to form nitrosamides (HN₂O₂), and N₂O₃ can react with amines to form nitrosamines (H₂N₂O). Both nitrosamides and nitrosamines are N-nitroso compounds and potentially carcinogenic. Vitamin C (C₆H₈O₆) can inhibit the nitrosation process, because it reacts faster than the amine with N₂O₃. Vitamin C reduces 2HNO₂ to NO, and is itself oxidized to dehydroascorbic acid. This will reduce the amount of N-nitroso compounds that can be formed.

Figure 2

Average values for creatinine corrected ATNC levels in urine (nmol/mmol) for the intervention groups stratified for vitamin C supplementation, at baseline (day 1), after consumption of 1 bottle (day 2) and 7 bottles (1 daily, day 8) of beetroot juice. Error bars indicate standard error. (a) p < 0.05; significant increase at day 8 compared to day 2 in the BRJ group (Wilcoxon signed rank test); (b) p < 0.01: significant increase at day 2 compared with baseline levels (day 1) in the BRJ group (Wilcoxon signed rank test); (c) p < 0.001; significant increase at day 8 compared to baseline levels (day 1) in the BRJ group (Wilcoxon signed rank test); (d) p < 0.01, significantly higher levels in the BRJ group compared to the BRJ + vit C group (Mann–Whitney test) at day 2; and (e) p < 0.01, significantly increased levels at day 8 in the BRJ + vit C group compared to baseline levels (day 1) and day 2 (Wilcoxon signed rank test).

Figure 3

Average values (± SE) for creatinine corrected nitrate levels in urine for the intervention groups stratified for vitamin C supplementation at baseline (day 1), after consumption of 1 bottle (day 2) and 7 bottles (1 daily, day 8) of beetroot juice. Wilcoxon signed rank test; significant increase compared with baseline. (a) p < 0.001, significant increase at day 2 as compared to baseline levels (day 1) in the BRJ group; (b) p < 0.001, significant increase at day 2 as compared to baseline levels (day 1) in the BRJ + VitC group; (c) p < 0.001, significant increase at day 8 as compared to baseline levels (day 1) in the BRJ group; (d) p < 0.001, significant increase at day 8 as compared to baseline levels (day 1) in the BRJ + VitC group.

Figure 4

Outline of the experimental protocol. The 29 study subjects were first randomized by block randomization. Sixteen subjects were attributed to the BRJ group and 13 to the BRJ + VitC group. On day 1, the subjects collected morning urine, and the baseline measurements (resting blood pressure and body mass index) were performed. In the evenings of days 1 to 7, the BRJ group ingested only beetroot juice (BRJ), while the BRJ + VitC group ingested both BRJ and vitamin C (BRJ+VitC). On day 2 and 8, all subject collected a morning urine sample. On day 8, the resting blood pressure and body mass index were measured again.