Salimetrics will be available at the British Psychological Society Conference on Stand E3 to discuss your research in more detail.
If you are new to the use of Saliva as a minimally invasive tool, please read the following Introduction to Saliva Research as used by many European University Psychology Departments:
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In 1992 Dr. Irwin Mandel addressed the first New York Academy of Sciences meeting on the topic of Saliva as a Diagnostic Fluid, where he referred to the concept of using saliva as “a mirror of the body.” At that time, the number of researchers who shared Dr. Mandel’s enthusiasm for saliva testing was relatively small, and there were some who felt that saliva could not serve as a reliable testing fluid. After nearly 20 years of subsequent work in this field, however, we are now witnessing a sharp rise in the acceptance of saliva as a valuable testing medium alongside, or as an alternative to, traditional testing fluids like blood and urine. This adoption of saliva has been especially evident in psychology and related fields, where researchers were among the first to recognise the advantages of multiple, non-invasive collections that saliva testing has to offer.
Saliva composition
Whole saliva is a complex mixture of water, ions and locally secreted organic compounds. Measurements of the locally secreted compounds in saliva, such as secretory immunoglobulin A (SIgA) and alpha-amylase (sAA), provide useful information on the biologically active portion of those analytes. In addition to locally secreted compounds, drugs, drug by-products, hormones and some proteins pass into saliva from the capillaries that surround the salivary glands. For neutral steroids that are able to passively diffuse across the salivary acinar cells, such as cortisol and testosterone, saliva concentrations correlate very highly with serum levels. This makes saliva testing a safer, non-invasive alternative to blood sampling
What can be measured?
Cortisol is the major glucocorticoid hormone produced in the adrenal cortex and is actively involved in the regulation of many physiological systems. Salivary cortisol has been studied extensively in research on human behaviour, emotions, and development; examples include studies involving anxiety, depression, PTSD, and behavioural disorders (Schlotz, 2006; Wessa, 2006; Gordis, 2006; Dorn 2009)
Due to their common origin as products of the HPA axis, DHEA and DHEA-S are often examined in conjunction with cortisol. Examples of studies that have measured salivary DHEA/DHEA-S have involved disruptive behaviour (Dorn, 2009) and depression (Assies, 2004). Given the important neuro-protective effects that have been recognised for DHEA and DHEA-S (Manninger, 2009), it seems likely that these two steroids will continue to be scrutinised in relation to brain health and mental disorders.
Psychology-related studies involving salivary steroids controlled by the HPG axis (estradiol, estrone, estriol, progesterone, and testosterone) have also grown significantly. Many studies of the sex hormones have focused on their roles in sexual function and fertility, and in these fields blood measures of these hormones are often still preferred. However, the sex hormones also have well-established links to emotions, behaviour, and development; and it is in these research areas that investigators have been quicker to exploit the advantages of salivary hormone testing (Stanton, 2009; Welling, 2007).
In addition to the steroid hormones, other small, neutral molecules such as melatonin also diffuse readily from blood into saliva. Salivary melatonin has excellent correlation with serum values, and a growing number of studies have been exploring it in connection with various disorders that may be related to light cycles and sleep (Novakova, 2011; de Almeida, 2011).
The digestive enzyme alpha-amylase (sAA), which is one of the major proteins secreted by the salivary glands, has been one of the analytes most heavily studied in connection with the nervous control of protein secretion. By its use as a marker of autonomic activity, research has revealed that sAA measurements are related to a variety of behavioural, social, health, and cognitive phenomena in human subjects (Nater & Rohleder, 2009). Many psychobiological studies now routinely include measurements of both cortisol and sAA in order to observe activity in the HPA axis and the ANS, respectively (Gordis, 2006; Vigil, 2010).
Another major protein found in saliva is secretory immunoglobulin A (SIgA). This important component of the immune system is different from most other salivary proteins in that it is neither synthesised by the salivary glands, nor related to IgA levels in the circulation. Rather, IgA that originates from B lymphocyte cells adjacent to the salivary glands is bound and actively transported through the salivary cells, then released into saliva as SIgA. (See figure 1.) Although the details of the control of secretion of SIgA into saliva are still not totally clear, it is believed that both the synthesis of IgA by the immune cells and its transportation into saliva are affected by ANS signals (Bishop, 2009). Consequently, SIgA is another biomarker whose levels in saliva have been found to change in response to various types of stress and mood states (Kugler, 1992).
Methodology
Investigators who intend to use saliva testing should understand that the methods used to collect and handle saliva samples can have a direct effect on their results.
Saliva can be collected by a number of methods; the most appropriate will depend on the analytes of interest and the age of the participants. For the Passive drool method, participants allow saliva to pool in the mouth before passing it through a small straw into a cryovial; this can be used for all analytes. When measuring sAA, cortisol, CRP, S-IgA or testosterone, participants unwilling or unable to drool into a vial may find an oral swab easier to use. A small, absorbent and non-toxic insert is placed in the mouth for 1 to 2 minutes before being placed in a storage tube. Child, infant and animal swabs are also available – designed to be held under the tongue to reduce choking hazard.
ELISA assays require very small volumes of saliva (10-100µL). While it is advisable to collect an extra 300µL to cover for liquid handling losses, in most cases collecting 0.5mL of saliva by the passive drool method, or 1 to 2 minutes of collection with an oral swab, is sufficient.
As steroid hormones are non-polar molecules, they have a tendency to be attracted to some types of plastic. Therefore it is important to select tubes or vials made from high-grade polypropylene to avoid retention of the analyte.
In preparation for testing, participants should avoid alcohol for 12 h prior to collection and food 1 h prior to collection; it may also be pertinent to record physical activity levels 24 h prior. Participants should rinse out their mouths with water 10 minutes prior to collection to remove any food residues.
Some analytes, such as cortisol, fluctuate markedly during a 24 h period according to a regular diurnal cycle, meaning the time of day that samples are collected should be considered; while others are affected by stimuli such as stress, which may need to be controlled. As S-IgA, DHEA-S and sAA are affected by flow rate, the time taken to collect the sample should be recorded and the quantity weighed; results can then be expressed as secretion rates (µg/min) rather than concentrations (mg/l).
Bacteria are present in saliva, and unless steps are taken to slow or stop their action, they can cause degradation of the analytes. Temperature stability varies between analytes; however, researchers are advised to freeze samples as soon as possible after collection. Samples can be stored at 4°C for a maximum of 4 hours, before freezing at -80°C for long term storage. Repeated freeze-thaw cycles should also be avoided, when investigating multiple analytes it is advisable to divide samples into smaller aliquots before freezing.
On commencing analysis, all samples should be thawed, vortexed and centrifuged to help break up any mucus and mix the samples. This will leave a clear solution, free from any unwanted particles, ready for use in the immunoassay.
Most immunoassays share to basic steps:
1. Pre-prepared antibodies (highly specific against the antigen/analyte of interest) are used to capture molecules of the antigen present in the sample, binding them to the microplate
2. A measurable label (conjugate enzyme) which attaches to the analyte is added, indicating the analytes presence by colour change
Once appropriate volumes of saliva sample and conjugate have been added to the microplate, and stated incubation periods and wash cycles observed, colour change can be read by a microplate reader. The amount of colour, or optical density, can then be compared using computer software to the range of known standard concentrations supplied with the kit; allowing the conversion of optical density values into concentrations of the analyte of interest.
What questions can be answered?
As demonstrated by the selected highlights from the literature given above, salivary biomarkers can be used to answer a wide range of questions within psychology. Perhaps most pertinent for those who are new to saliva testing, it can offer an objective and fully quantitative result to assess psychological disorders, such as stress, anxiety and depression. Testing can be incorporated into longitudinal study designs to investigate the progression of these disorders, overcoming some of the issues that surround the use of questionnaires and participant recall. While cross-sectional designs can be employed to identify potential physiological ‘risk factors’ may be particular conditions.
Saliva testing is also frequently used to assess the physiological response to psychological stimuli, both in lab and field-based scenarios. Concentrations of salivary biomarkers change within minutes in response to psychological stimuli, allowing researchers to analyse a participant’s response to even the most acute stimuli.
The simple and safe nature of sample collection lends itself to studies that require measurements to be taken ‘in the field’, for example those examining sleep patterns or the cortisol-awakening response. It also represents a less stressful alternative to blood sampling, which may be of importance for those evaluating stress, anxiety and depression.
What’s new?
An additional related topic receiving considerable attention is the relationship between systemic inflammation and brain health. It has been demonstrated that physical and mental stress lead to increased levels of circulating pro-inflammatory cytokines, such as IL-6, IL-1b, and TNF-a, which in turn are thought to interact with the brain; contributing to the development of mental disorders such as major depression (Debnath, 2011).
Salivary cytokines also appear to be influenced by the same outflow of nervous signals that affect systemic levels, and a small number of papers have observed that these salivary markers vary in connection with stress and various other mental conditions (Keller, 2010; Sjögren, 2006). This area of research is relatively new, and the details of the control of these salivary markers are not yet well understood; studies to date have found that the correlation between salivary and serum levels is only modest (Sjögren 2006). Further work is therefore needed to assess more fully the significance and general utility of these salivary markers of inflammation for psychobiological research.
Hair has long been analysed for exogenous compounds, specifically drugs of abuse, providing a useful tool in detecting long-term drug exposure. More recently, evidence has shown that hormones become trapped in hair as it grows, opening the potential for cortisol analysis as a quantitative measure of chronic stress (Gow, 2010). While emerging evidence is encouraging (Kirschbaum, 2009; Thomson, 2010), further work is required to validate the use this method in psychology.
References
Assies , J., Visser, I., Nicolson, N.A., Eggelte, T.A., Wekking, E.M., Huyser, J., Lieverse, R., & Schene, A.H. (2004). Elevated salivary dehydroepiandrosterone-sulfate but normal cortisol levels in medicated depressed patients: Preliminary findings. Psychiatry Res, 128(2), 117-22.
Bishop, N.C. & Gleeson, M. (2009). Acute and chronic effect of exercise on markers of mucosal immunity. Front Biosci, 14, 4444-56.
De Almeida, E.A., Di Mascio, P., Harumi, T., Spence, D.W., Moscovitch, A., Hardeland, R., Cardinali, D.P., et al. (2011). Measurement of melatonin in body fluids: Standards, protocols and procedures. Childs Nerv Syst, 27, 879-91.
Debnath, M., Doyle, K.M., Langan, C., McDonald, C., Leonard, B., & Cannon, D.M. (2011). Recent advances in psychoneuroimmunology: Inflammation in psychiatric disorders. Transl Neurosci, 2(2), 121-37.
Dorn, L.D., Kolko, D.J., Susman, E.J., Huang, B., Stein, H., Music, E., & Bukstein, O.G. (2009). Salivary gonadal and adrenal hormone differences in boys and girls with and without disruptive behavior disorders: Contextual variants. Biol Psychol, 81(1), 31-39.
Gordis, E.B., Granger, D.A., Susman, E.J., & Trickett, P.K. (2006). Asymmetry between salivary cortisol and α-amylase reactivity to stress: Relation to aggressive behavior in adolescents. Psychoneuroendocrinology, 31(8), 976-87.
Gow, R., Thomson, S., Rieder, M., Van Uum, S., & Koren, G. (2010). An assessment of cortisol analysis in hair and its clinical applications. Forensic Sci Int., 196, 32-37
Keller, P.S., El-Sheikh, M., Vaugh, B., & Granger, D.A. (2010). Relations between mucosal immunity and children’s mental health: The role of child sex. Physiol Behav, 101(5), 705-12.
Kirschbaum, C., Tietze, A., Skoluda, N., & Dettenborn, L. (2009). Hair as a retrospective calendar of cortisol production – Increased cortisol incorporation into hair in third trimester pregnancy. Psychoneuroendocrinology, 34, 32-37.
Kugler, J., Hess, M., & Haake, D. (1992). Secretion of salivary Immunoglobulin A in relation to age, saliva flow, mood states, secretion of albumin, cortisol and catecholamines in saliva. J Clin Immunol, 12(1), 45-49.
Maninger, N., Wolkowitz, O.M., Reus, V.I., Epel, E.S., & Mellon, S.H. (2009). Neurobiological and neuropsychiatric effects of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Front Neuroendocrinol, 30(1), 65-91.
Nater, U.M. & Rohleder, N. (2009). Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: Current state of research. Psychoneuroendocrinology, 35(10), 1565-72.
Nováková, M., Paclt, I., Ptácek, R., Kuželová, H., Hájek, I., & Sumová, A. (2011). Salivary melatonin rhythm as a marker of the circadian system in healthy children and those with attention-deficit/hyperactivity disorder. Chronobiol Int, 28(7), 630-7.
Schlotz, W., Hellhammer, J., Schulz, P., & Stone, A.A. (2006). Perceived work overload and chronic worrying predict weekend-weekday differences in the cortisol awakening response. Psychosom Med, 66(2), 207-14.
Sjögren, E., Leanderson, P., Kristenson, M., & Ernerudh, J. (2006). Interleukin-6 levels in relation to psychosocial factors: Studies on serum, saliva, and in vitro production by blood mononuclear cells. Brain Behav Immun, 20(3), 270-78.
Stanton, S.J. & Schultheiss, O.C. (2009). The hormonal correlates of implicit power motivation. J Res Pers, 43(5), 942-49.
Thomson, S., Koren, G., Fraser, L.A., Rieder, M., Friedman, T.C., & Van Uum, S.H. (2010). Hair analysis provides a historical record of cortisol levels in Cushing’s syndrome. Ex Clin Endocrinol Diabetes, 118, 133-38
Vigil, J.M., Geary, D.C., Granger, D.A., & Flinn, M.V. (2010). Sex differences in salivary cortisol, alpha-amylase, and psychological functioning following Hurricane Katrina. Child Dev, 81(4), 1228-40.
Welling, L.L., Jones, B.C., DeBruine, L.M., Conway, C.A., Smith, M.J., Little, A.C., et al. (2007). Raised salivary testosterone in women is associated with increased attraction to masculine faces. Horm Behav, 52(2), 156-61.
Wessa, M., Rohleder, N., Kirschbaum, C., & Flor, H. (2006). Altered cortisol awakening response in posttraumatic stress disorder. Psychoneuroendocrinology, 31(2), 209-15.
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