Detection of nitrate and nitrite in human saliva and other biofluids after nitrate ingestion, and optimisation of an electrochemical sensor for salivary nitrite

Abstract

Drugs such as proton pump inhibitors and broad-spectrum antibiotics may affect the integrity of the human enterosalivary circulation of ingested nitrate. Here, this idea was tested, alongside experiments to optimise and validate a commercial electrochemical potentiostat sensor for measuring salivary nitrite.

Method validation of a spectrophotometric (Griess) assay and an ozone-based chemiluminescence assay demonstrated that both were suitable for measuring nitrate/nitrite in gastric juice, serum and saliva in five healthy H.pylori negative volunteers who had received short courses of proton pump inhibitor (omeprazole), an antibiotic (co-amoxiclav) or neither of these drugs, in each case followed by an oral nitrate bolus. Omeprazole increased the intragastric pH following oral nitrate ingestion (e.g., at 30 min, mean pH=6.5; p<0.0001), compared with the same volunteers who received oral nitrate alone. Omeprazole decreased the mean serum nitrate concentration at 90 min (154 μmol/L; p=0.0312) after potassium nitrate ingestion. Omeprazole may decrease the chemical reduction of intragastric nitrite to nitric oxide by increasing the local intragastric pH. Following co-amoxiclav treatment and nitrate ingestion, the mean salivary nitrite concentration was lower at 120 min (163 μmol/L; p=0.007) compared with volunteers who received oral nitrate alone. This may be explained by co-amoxiclav having destroyed the oral nitrate-reductase bacteria. The correlation between serum nitrate and salivary nitrate was maintained (r>0.97) when the volunteers had either no drug (n=20), omeprazole (n=16), or co-amoxiclav (n=20). Salivary nitrite correlated with serum nitrate (r=0.90) and salivary nitrate (r=0.94) when the volunteers had no drug (n=20), suggesting that salivary nitrite could be a surrogate marker for serum nitrate. Furthermore, significant correlation was observed between serum nitrite and salivary nitrite (r=0.89) when the volunteers had no drug (n=20).

The electrochemical sensor for salivary nitrite was linear from 0-2000 μmol/L, had an intra-assay coefficient of variation <20% and was accurate except when saliva was spiked with 200 μmol/L nitrite. When twenty saliva samples from healthy volunteers were subjected to one freeze-thaw cycle, a mean bias of 1382 μmol/L was observed between the sensor (mean 1496 μmol/L) and the Griess method (114 μmol/L). However, when saliva samples (n=8) underwent ≥3 freeze-thaw cycles, followed by spiking with nitrite, the bias between the two methods decreased to 607 μmol/L, and the correlation between the two methods increased (r=0.85; p=0.0008). Nonetheless, a proportional and constant error was noted between the two methods.

Omeprazole treatment, followed by a nitrate bolus, increased the intragastric pH and decreased serum nitrate compared with nitrate ingestion alone. Co-amoxiclav affected the reduction of oral nitrate to nitrite as demonstrated by the reduction in salivary nitrite. Square wave voltammetry is a potential method for the clinical measurement of salivary nitrite.