Validity and reliability of test strips for the measurement of salivary nitrite concentration with and without the use of mouthwash in healthy adults

The nitrate (NO3-)-nitrite (NO2-)-nitric oxide (NO) pathway has received considerable interest in recent years as a potential target for nutritional interventions designed to increase NO production, and elicit therapeutic effects in humans. In particular, studies have evaluated the effects of supplemental dietary NO3-, which serves as a 'substrate' for this pathway, on numerous different health outcomes. One challenge has been to evaluate compliance with the NO3- interventions. A recent advance in this field has been the development of a non-invasive, simple and rapid method to measure nitrite concentrations in saliva using small test salivary strips. In the present study, ten healthy adults were recruited to a randomised, crossover study and received an acute dose of NO3- -rich beetroot juice (BJ) after rinsing their mouth with either water or commercially available antibacterial mouthwash. Salivary NO3- and NO2- concentrations were measured at baseline and up to 5 h after BJ consumption using the gold-standard chemiluminescence and a colorimetric Griess assay. In addition, two salivary test strips (Berkeley Test strips, CA, USA) were used to measure NO(2)(-)concentrations at the same time points. Five observers read the strips and inter- and intra-observer reliability was measured. The Bland-Altman method was used to provide a visual representation of the agreement between the methods used to evaluate salivary NO3-/NO(2)(-)concentration. Sialin concentrations were measured at baseline and up to 5h after BJ consumption. BJ elevated salivary NO3- and NO2- concentrations when the mouth was rinsed with water (both P < 0.01), as assessed via both chemiluminescence and Griess methods. Rinsing the mouth with antibacterial mouthwash attenuated markedly the increase in NO2- (P < 0.001), while NO(3)(-)concentrations were unaffected (P > 0.05). The Intra-Class Coefficients of Correlation (ICC) showed a high inter- and intra-observer reliability (r > 0.8). A significant positive correlation was found between absolute salivary NO2- concentrations measured by strips and Griess and chemiluminescence methods (rho = 0.83 and 0.77, respectively) and also when expressed as changes in salivary NO2- concentrations (rho = 0.80 and 0.79, respectively). Bland Altman analysis indicated a poor agreement for absolute NO2- concentrations between salivary strips and the chemiluminescence and Griess methods. A small significant negative correlation was found between changes in salivary sialin and salivary NO2- concentrations (r = -0.20, P = 0.04). A non-significant positive correlation was observed between the change in salivary sialin and salivary NO3- concentrations (r = 0.18, P = 0.06). This study suggests that commercially available salivary NO2- test strips provide a reasonable surrogate marker for monitoring changes in salivary NO2- concentrations in humans. However, the strips do not provide accurate estimates of absolute NO2- concentrations.