Salivary Redox Status, Main Antioxidants and Methods for Characterizing it

Saliva plays important role in bodily protection → Protection involves secretion of salivary proteins that regulate production of ROS and RNS → ROS and RNS are toxic because they react with biomolecules.

Production of antioxidant compounds to regulate reactive species is essential.

Antioxidant capacity of saliva has been suggested to modulate the metabolization of flavor compounds → Impact on release and perception of flavors.

Salivary Redox Status

Reactive Oxygen Species

Molecular dioxygen can lead to formation of reactive species → lead to formation of ROS

ROS:

very reactive with very short lifespan
generated in oral epithelium and directly in saliva
regulate the oral microbiota

Deregulation of saliva redox balance → Good diagnostic indicator for pathologies not directly linked to oral cavity.

Superoxide anion (O2·−)
- Precursor of many ROS
- Can lead to the generation of the hydroxyl radical (OH·)
Hydroxyl radical (OH·)
- Produced via Haber-Weiss reaction between superoxide anion and hydrogen peroxide
- Produced via Fenton reaction between reduced iron (Fe2+) and hydrogen peroxide
Hydrogen peroxide (H2O2)
- Results from dismutation of superoxide anion
  - Its production can be catalyzed by superoxide dismutase (enzyme found in saliva)
- Can be produced and released in saliva by oral microbiota
- Particularly toxic because it can produce the hydroxyl radical (OH·) by reacting direcly with superoxide anion ithout requiring the Fenton reaction

Salivary Antioxidant Capacity

Fig. 1 Schwartz et al., Frontiers in Nutrition, 2021

Elimination of ROS

Main neutralizing enzymes:

Superoxide dismutases (SOD)
- Catalyze the dismutation of two O2.- molecules into H2O2 → H2O2 can be eliminated by the catalase or peroxidase systems
- Directly remove superoxide anion
Catalase
- Allow dismutation of H2O2 into H2O and O2
Peroxidases
- Hydrogen peroxide reduction
- Antimicrobial potential → Oxidation of thiocianate ions

Low-molecular weight molecules:

Uric acid
- Main antioxidant molecule in human saliva
- Responsible for 70% of antioxidant activity of saliva
- Its concentration in saliva correlates with that of plasma
Ascorbic acid (Vitamin C)
Tocopherols (Vitamin E)
Glutathione
- Can act directly on ROS
Thioredoxin
- Can directly neutralize certain ROS (hydroxyl radical and singlet oxygen)
Histatins

Methodologies for characterizing salivary redox status

Two approaches:

Measure oxidative stress biomarkers
Measure the total antioxidant capacity → Global measurement of the amount of reducing compounds

Total antioxidant capacity: sum of the antioxidant molecule activities in saliva

Methods:

Ferric-reducing antioxidant power (FRAP) test
Amount of uric acid
Antioxidant capacity measurements in Trolox equivalent (TEAC)
- Evaluates the combined action of the different free radical scavengers present in saliva

Pre-analytical considerations:

Common practice is centrifuging saliva before storing at -20°C
- Some salivary enzyme activity can be lost → Reduction in inter-individual differences
- Each protein eliminated contains cysteine residues → Contributes to FRAP and TEAC indices
Oxidation of the sample by air oxygen presents a major bias

Preprint

Preprint

Salivary Redox Status, Main Antioxidants and Methods for Characterizing it

Salivary Redox Status, Main Antioxidants and Methods for Characterizing it