Supplementary Material for: Adding Nitrate to a Sucrose Rinse Reduces Acidogenicity: An In Vivo Study

BACKGROUND: Potential dental health benefits, including pH buffering, have been demonstrated for nitrate in placebo-controlled studies when consumed one or several hours before sugar intake. However, its immediate local effect when co-administered with sugar is unclear.
AIM: To test the local effect of adding nitrate to a sugar rinse on human saliva acidification.
METHODS: A double-blind, crossover study was conducted with three periods. In each period, 18 adults, equally distributed between men and women (n=9 each), aged 25.4±3.8 (mean±SD), with normal salivary flow, rinsed for 2-min using 10-mL of one of the following rinses: spinach-only (blended spinach providing 8-mM nitrate), spinach+sucrose (blended spinach providing 8mM nitrate + 20% sucrose), or sucrose-only (20% sucrose). Unstimulated saliva was collected at baseline and every 3-min up to 24-min. pH, lactate, nitrate, and nitrite were measured using Reflectoquant®. The effect of treatment and time was assessed using a linear mixed model (α=0.05). Nitrate reduction capacity was determined in vitro using baseline saliva.
RESULTS: All saliva samples demonstrated nitrate reduction activity. The lowest pH values were observed 6-min after rinsing, with all groups differing from each other: sucrose-only (6.56±0.38; mean±SD), spinach+sucrose (6.83±0.37), spinach-only (7.15±0.44, no pH drop). The pH returned to baseline values 12-min after the spinach+sucrose rinse, but not after the sucrose-only rinse at any time point. Nitrate and nitrite concentrations increased in saliva after using spinach-containing rinses, returning to their baseline levels after 6-min and 12-min. Lower lactate levels were detected after 12-min, suggesting proton consumption during nitrate metabolism in the spinach+sucrose group.
CONCLUSION: Adding a low dose of nitrate to a sucrose rinse limited sugar-induced salivary acidification in vivo, supporting the potential for topical nitrate strategies to reduce sugar acidogenicity.