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Saliva as a Testing Fluid

Saliva gaining momentum as an effective testing fluid for research, diagnostic applications

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In recent years saliva has increasingly gained recognition as an important testing fluid that can stand alongside blood and urine. Saliva samples can be collected in a non-invasive, stress-free manner without needles, and its collection has no issue of personal privacy that comes with collecting urine samples. As well, saliva is ideal for use in studies that involve children or that require collection of multiple samples to track biomarker rhythms or monitor stress reactivity.

It is now widely recognized that saliva contains many of the same biomarkers commonly measured in other testing fluids. Small, neutral molecules such as the steroid hormones, melatonin and various drugs or drug metabolites (cotinine) diffuse easily from blood into saliva. Some molecules that are too large or too electrically charged to enter by passive diffusion can still find their way into saliva by alternate mechanisms, including active transport (secretory IgA, insulin) or ultrafiltration (DHEA sulfate).

Proteins of Interest
Some large blood proteins such as albumin or binding globulins are too big to pass into saliva through any of the above processes unless oral tissues are compromised by inflammation or injury. Very low levels of larger blood proteins can still be found in saliva of healthy individuals, however, and it is believed that these molecules enter saliva principally through the outflow of the serum-like gingival crevicular fluid (GCF) from the gingival tissues surrounding the teeth. The acute phase protein CRP is a good example of a commonly measured serum protein that can be found in low levels in saliva.

Researchers are examining the relationship between circulating and salivary levels of CRP, and preliminary evidence has suggested that salivary CRP can discriminate between high and low serum CRP levels and, therefore, has potential to serve as a screen for CVD risk status.1

Because many steroid hormones in the bloodstream are bound to albumin or to specific hormone binding globulins, the numbers of free molecules available for diffusion into saliva may be relatively small. Salivary levels of steroids such as cortisol, estradiol and testosterone, for example, are only 1-10% of those found in serum. Modern immunoassays have sufficiently good sensitivity to work in these ranges, however, so these low levels are generally not a problem. In fact, the ability to measure free hormone levels directly through saliva is regarded as an advantage, and salivary cortisol has been shown to be preferable to serum cortisol for determination of adrenocortical function.

IgG and the monomeric form of IgA also leak into saliva from blood through GCF. They are present in much lower levels than dimeric IgA and pentameric IgM, however, which are secreted by immune cells adjacent to the salivary glands, then actively transported into saliva as secretory IgA (SIgA) and secretory IgM (SIgM). Salivary SIgA has often been utilized as a marker of mucosal immunity and studied in conjunction with stress markers such as cortisol.

In addition to the proteins that enter saliva from outside the salivary glands, numerous peptides and proteins are released into the oral cavity from the salivary glands (and related nerves), various oral tissues, and microbes. More than 3,000 proteins have now been catalogued in saliva, and work is under way to understand their functions and potential uses as biomarkers.

Examples of proteins being studied for their relationships to inflammation, immune function and cell regeneration include cytokines (IL-1β, IL-6, TNF-α), neuropeptides (VIP, NPY) and growth factors (EGF, NGF, BDNF). These types of protein molecules are of obvious interest to dental researchers and oral biologists, but they are also increasingly being scrutinized by researchers in other fields for potential use as biomarkers related to health and disease outside the oral cavity.

A major salivary protein of particular interest is the enzyme alpha-amylase (sAA), which serves digestive and anti-microbial roles in the mouth. Because salivary proteins are released in response to signaling from the sympathetic and parasympathetic nervous systems, researchers are studying salivary proteins such as sAA as markers of activity in the autonomic nervous system (ANS). Traditional ANS assessment methods such as monitoring changes in skin conductance or heart function require specialized instrumentation and are not practical for many types of research projects. The ability to assess ANS activity through these salivary markers such as sAA is, therefore, a significant scientific advance, since it allows researchers to investigate ANS activity along with other related biomarkers in more naturalistic settings.

Due to their local synthesis and release into the oral cavity, levels of salivary protein markers may correlate only modestly or weakly with circulating levels of the same markers. Nevertheless, research is beginning to show that protein markers in saliva may have responses to stress or inflammation that parallel those found in other compartments, which may allow them to serve as useful biomarkers in certain diagnostic and research applications. Additionally, many salivary proteins are unique to saliva, and some may have the potential to provide information that cannot be obtained from other testing fluids.
 
Nucleic Acids
In addition to containing many proteins of interest, saliva also contains nucleic acids. It has recently been demonstrated that saliva is a convenient source of high-quality DNA that can be used in genotyping studies,2 which makes possible genetic and biomarker analysis from a common saliva sample. Various mRNAs and micro-RNAs have also been identified in saliva, and some have already shown value as markers of oral and non-oral cancers.

In the Know
Given the complex physiological factors that affect biomarker levels in saliva, researchers new to saliva testing need to familiarize themselves with issues that can affect the quality of their testing results.  These factors include location in the mouth where samples are collected, changes in saliva flow rate, diurnal rhythms of salivary analytes, presence of blood contamination or oral inflammatory diseases, and proper timing of sample collection to capture responses to stressors or awakening responses.
  
Additionally, working with saliva in the lab presents some challenges not found in other testing fluids. Because of the presence of mucoproteins, saliva is a sticky, viscous fluid that can be difficult to pipette with accuracy.  Saliva also contains substances that can cause interference in immunoassays. It is important to prepare samples properly and use immunoassays that have been verified for use with saliva.

Saliva contains a variety of enzymes from glands, tissues and microbes that can significantly degrade many analytes, and some markers have poor freeze-thaw stability. Proper collection, storage and handling procedures for all analytes involved in a study must therefore be understood prior to beginning the project.

Saliva is a valuable testing medium that makes possible non-invasive, ecologically valid testing of key biomarkers that represent diverse systems within the body. It contains many of the same analytes that are traditionally measured in blood or urine, as well as some unique to saliva. Although saliva is not able to duplicate all the uses of other testing fluids, it does have many practical advantages and combinations of analytes that may make it the fluid of choice for many research and diagnostic applications.

Doug Maple is technical publications associate, Salimetrics, and Dr. Granger is professor of Nursing, Public Health and Medicine, and director, Center for Interdisciplinary Salivary Bioscience Research at The Johns Hopkins University. Dr. Granger is also founder and chief scientific and strategy advisor of Salimetrics.

References
1. Out, D., Hall, R.J., Granger, D.A., Page, G.G., & Woods, S.J. (2012). Assessing salivary C-reactive protein: Longitudinal associations with systemic inflammation and cardiovascular disease risk in women exposed to intimate partner violence. Brain Behav Immun, 26(4), 543-51.

2. Nemoda, Z., Horvat-Gordon, M., Fortunato, C.K., Beltzer, E.K., Scholl, J.L., & Granger, D.A. (2011). Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples. BMC Med Res Methodol, 11:170.



     

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