What is saliva and why is it important for diagnostics?

Aside from when we lick a stamp, get a whiff of greasy fast food, or hold our mouths open for a routine dental cleaning, we typically don’t give saliva more than a fleeting thought. In the diagnostics world, the same has been historically true: Oral sample types, like saliva, have been ignored and undervalued, reserved only for tests for oral infections or cancers.¹

But over the past decade, there’s been a major sea change in salivary diagnostics and saliva has quickly risen as a sample type with huge potential beyond applications in oral health. Look no further than 23and Me’s FDA-approved, direct-to-consumer (DTC) Genetic Health Risk test for proof.² The test is the first of its kind and determines an individual’s risk of developing 10 medical diseases or conditions, all with DNA extracted from a simple saliva sample.

The advantages of saliva as a diagnostic sample type

It’s easy to see why saliva is entering the fold as an alluring diagnostic sample type: It’s inexpensive to collect, can be sampled without the help of a medical professional and with minimal subject discomfort, and has been successful for human health monitoring, disease surveillance for the ongoing SARS-CoV-2 pandemic, and human microbiome analysis.^3-6 Despite its high water content (~99%), it’s also an incredibly complex biofluid with interesting physical and chemical properties and plays a critical role in normal human physiology and disease pathophysiology. 

To gain a better appreciation for saliva and its growing use within the diagnostics market, we’ll take a closer look at some foundational questions about saliva’s composition, physiological function, and its under-appreciated history in the medical field.

Taking a “lick” at composition: What is saliva made of?

Saliva is a complex mixture derived from the secretions of several different types of salivary glands. The composition and properties of these secretions make many normal, everyday functions possible and vary due to the time of day, diet, age, gender, disease states, or administration of pharmacological agents.⁸

90% of the total salivary volume is made of the secretions of 3 glands, collectively called the major salivary glands: the parotid, the submandibular, and the sublingual glands.⁸ Whole saliva, which fills our mouths daily at the astounding volume of 1 to 1.5 liters, contains both inorganic and organic components, secreted not only from the major glands, but also the secretions of the minor glands, gingival crevicular fluid, and much more.^8-10

Inorganic components of saliva

The inorganic components of whole saliva include Na+, Cl– Ca2+, K+, HCO3–, H2PO4–, F–, I–, Mg2+ and thiocyanate.^9,10 Calcium and phosphate help to neutralize acids and therefore, protect against tooth decay. Bicarbonate also acts as a buffer, maintaining the pH of saliva slightly acidic, between 6 and 7.^9,10

Organic components of saliva

There is a much larger and more diverse collection of organic components that make up saliva. Organic compounds include urea, ammonia, glucose, free fatty acids, triglycerides, amino acids, and many others.

There are also a substantial number of key proteins including mucins, amylases, agglutinins, glycoproteins, lysozymes, peroxidases, lactoferrin, immunoglobulins, lactoferrin, myeloperoxidase, histatins, cystatins, statherins, and defensins, all of which help perform a variety of salivary functions.^9,10 

To give these inorganic and organic electrolytes, proteins, and other compounds more context, let’s take a look at the overall functions of saliva and how each of these components fits in.

What is the function of saliva and what are its key components?

Each salivary component can be categorized as having one of five major functions (though some have multiple functions):

1. Lubrication and chemical/physical protection,
2. Buffering and clearance
3. Maintenance of tooth integrity
4. Immune activity and microbial protection
5. Taste and digestion.^7-11

Saliva provides oral lubrication and protection

As oral tissues are open to the exogenous environment, it’s under constant assault by toxic chemicals (i.e., carcinogens), microbes (i.e., beneficial and pathogenic), enzymes produced by plague, and desiccation from constant breathing.⁹ Mucins, specifically two highly studied glycoproteins called highly-glycosylated mucin (MG1) and single-glycosylated peptide chain mucin (MG2), play an important multi-functional role in the lubrication and protection of teeth and oral tissue.

Physical properties, such as high viscosity/elasticity/adhesiveness and low solubility, enable these and other mucins to lubricate tissue-tissue and tissue-teeth interactions, facilitating mastication, speech, and swallowing.⁹ Mucins also bind directly to tooth enamel, promoting the growth of beneficial bacteria, the clearance of caries-causing bacteria (i.e, Streptococcus mutans), acid resistance, and mineral stabilization at the tooth surface.⁹

Saliva for buffering and clearance

As mentioned above, bicarbonate plays a major role as a buffer, which neutralizes plaque-promoting acid.^9,12 Histidine-rich proteins (called histatins), urea, and phosphate also help buffer against significant pH change.^9,13 Salivary flow rate, which naturally varies due to many factors, is an additional mechanism by which major pH changes are avoided, promoting clearance of plaque-promoting bacteria and compounds.⁹

Saliva helps maintain tooth integrity

The inorganic and organic salivary components mentioned above help prevent demineralization of tooth enamel by buffering against large pH changes. Proteins such as statherins, histatins, cystatins, and several others form a protective barrier, called a pellicle, around tooth enamel and actively promote remineralization with calcium and phosphate.⁹ This enables long-term maintenance of protective tooth enamel.

Fluoride also helps promote the formation of stronger, more caries-resistant tooth enamel, by replacing magnesium and carbonate within enamel crystals.⁹

Saliva supports immune activity and microbial protection

The oral cavity can often be the first point of contact and a common route of transmission for pathogenic microbes, including viruses, bacteria, and fungi. Accordingly, one important function of saliva is as the first line of defense against microbial threats. 

This defense system consists of lysozyme, peroxidase, lactoferrin, immunoglobulins (i.e., IgA, IgG, and IgM), and a wide array of antimicrobial peptides that can recognize and/or attack specific microbial invaders.^7,9 These salivary components act directly on microbes with distinct mechanisms of action.^7,9

Other proteins, such as mucins, statherins, agglutinins, and histatins, prevent the growth of certain microbes in the oral cavity via a more indirect mechanism of action. These proteins promote aggregation and clearance of viruses, bacteria, and fungi preventing attachment and colonization.⁹

Saliva boosts taste and starts the digestion process

The final function of saliva we’ll discuss is perhaps the most top-of-mind for many – facilitating taste and beginning digestion. Saliva is hypotonic (i.e., it contains lower concentrations of certain ions than is present in plasma), enhancing the ability to taste salty foods.^7,9 Saliva also contains amylase and lipases, enabling preliminary digestion of starches and fats from food, as a food bolus is formed, swallowed, and delivered to the digestive tract.⁹ 

Over the past century, the composition of saliva and its physiological function has been heavily studied and clearly elucidated. Yet, as stated earlier, saliva is only now emerging as a powerful sample type for diagnostics, with approved DTC testing and successful use of salivary diagnostics as a “gold standard” for SARS-CoV-2 detection.^2,4

To better understand where the saliva testing field is going, we need to know where we’ve been. Let’s see how salivary diagnostics began and how it’s evolved over the past century.

The history of saliva diagnostics: From the Rice Test to today

Blood and urine found a rapid place in early medicine, with uroscopy (the visual examination of urine) dating as far back as the year 1090 in Jerusalem.^8,11 By contrast, saliva’s role in medicine came (relative to other biofluids) much later and it was instead used in an unexpected role as an ancient polygraph, called the “Rice Test.”^8,11 Several cultures used the “Rice Test” to assess if a person was guilty of an accused crime, administering a mouthful of dry rice to the accused. If anxiety, presumed to be heightened if one was guilty, prevented significant salivation and subsequent bolus formation, then the accused was deemed guilty.^8,11 

Saliva didn’t find its way into medical diagnostics until the early 1900s, where it was used as a very crude assessment of diathetic diseases (i.e. “abnormal states or conditions”), such as gout or migraine.^8,11 Since then, research has slowly ramped up and in the 1990s, Irwin D. Mandel, an early thought leader in salivary diagnostic testing, deemed that the field “…is a late bloomer, coming into flower as more people appreciate saliva as a mirror of the body…”^8,11 By this time the list of what exactly saliva helps mirror included tissue fluid levels, emotional status (from mania to depression), hormone levels, and more, opening up the potential for significant application to the development of diagnostics for systemic human diseases.^8,11

As a result, sensitive and specific salivary biomarkers have been identified for genetic diseases (i.e., cystic fibrosis), autoimmune disorders (i.e., Sjogren’s syndrome), cancer (i.e., oral squamous cell carcinoma), and have been particularly useful for drug monitoring (i.e., both recreational and prescribed).¹⁰

Saliva testing has also found another diagnostic home as a particularly favorable sample type for detecting infectious diseases. In the case of HIV, human herpes virus, cytomegalovirus, Epstein-Barr virus, and hepatitis C virus detection, saliva testing is more sensitive and specific than serum and urine testing.¹⁰ For bacterial pathogens, saliva has also been useful for the detection of Helicobacter pylori associated with peptic ulcers and S. mutans or lactobacilli associated with dental caries.^8,11

Advancing the promises of salivary diagnostics with SpeciMAX Dx Collection

The promise of saliva as an advantageous sample type is now being widely accepted. But to continue its advance, supporting products that promote streamlined, high-throughput sample collection, preparation, assaying, and analysis need to be developed.

See how to use the Thermo Scientific SpeciMAX Stabilized Saliva Collection Kit with this short protocol video.

To advance this cause, Thermo Fisher Scientific has developed the SpeciMAX Dx™ Collection Kits, designed for simplified self-collection of saliva samples for SARS-CoV-2 testing and beyond. The kits are optimized for automated liquid handling, nucleic acid extraction, and direct-to-PCR workflows and offer significant space, time, and cost savings compared to other collection kits.

If you’re interested in learning more, check out our other articles on 5 Benefits of Saliva Testing, or for Top 14 Tips for RNA Extraction.

 

This article is for Research Use Only. Not for use in diagnostic procedures.

 

References:

1. Malamud D. Saliva as a diagnostic fluid. Dent Clin North Am. 2011;55(1):159-178.
2. FDA allows marketing of first direct-to-consumer tests that provide genetic risk information for certain conditions. FDA website: https://www.fda.gov/news-events/press-announcements/fda-allows-marketing-first-direct-consumer-tests-provide-genetic-risk-information-certain-conditions. Published April 6th, 2017. Accessed September 20th, 2021.
3. Javaid MA, Ahmed AS, Durand R, Tran SD. Saliva as a diagnostic tool for oral and systemic diseases. J Oral Biol Craniofac Res. 2016;6(1):66-75.
4. Tan SH, Allicock O, Armstrong-Hough M, Wyllie AL. Saliva as a gold-standard sample for SARS-CoV-2 detection. Lancet Respir Med. 2021;9(6):562-564.
5. Saliva sampling using the SpeciMAX Saliva Collection Kit. Thermo Fisher Scientific website: https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/saliva-sampling-specimax-saliva-collection-kit-app-note.pdf. Published July 26th, 2021. Accessed September 20th, 2021.
6. Marotz CA, Sanders JG, Zuniga C, Zaramela LS, Knight R, Zengler K. Improving saliva shotgun metagenomics by chemical host DNA depletion. Microbiome. 2018;6(1):42.
7. Functions of Saliva. IntechOpen website: htps://www.intechopen.com/chapters/66233. Published October 23rd, 2019. Accessed September 17th, 2021.
8. Mandel ID. Salivary diagnosis: promises, promises. Ann N Y Acad Sci. 1993;694:1-10.
9. Humphrey SP, Williamson RT. A review of saliva: normal composition, flow, and function. J Prosthet Dent. 2001;85(2):162-169.
10. Greabu M, Battino M, Mohora M, et al. Saliva–a diagnostic window to the body, both in health and in disease. J Med Life. 2009;2(2):124-132.
11. Mandel ID. Salivary diagnosis: more than a lick and a promise. J Am Dent Assoc. 1993;124(1):85-87.
12. What Is Dental Plaque? Healthline website: https://www.healthline.com/health/dental-and-oral-health/plaque. Published August 2th, 2019. Accessed September 19th, 2021.
13. Mandel ID. The role of saliva in maintaining oral homeostasis. J Am Dent Assoc. 1989;119(2):298-304