Scientific References

1. The measurement of hormones in saliva: Possibilities and pitfalls Ross F. Vining, Robynne A. McGinley Garvan Institute of Medical Research, St Vincent’s Hospital, Sydney, NSW 2010, Australia Journal of Steroid Biochemistry Volume 27, Issues 1–3, 1987, Pages 81–94

Abstract

The easy stress-free, non-invasive nature of saliva collection makes it one of the most accessible body fluids and it is potentially of value in studying normal human physiology as well as pathology. Measurements of salivary hormone levels will usually only be of value if they reflect the plasma level of the hormone and the relationship between the saliva and plasma levels of many hormones have been studied by a number of groups. The measurement of the salivary level is a valuable clinical tool for some hormones (e.g. Cortisol, oestriol, progesterone), is of little value for others (e.g. cortisone, dehydroepiandrosterone sulphate, thyroxine, pituitary hormones) and for many others the saliva/plasma relationship is not yet sufficiently understood to assess the value of the salivary measurement. As well as reviewing the state of our knowledge of the salivary concentration of many hormones this review outlines a number of “rules of thumb” concerning the presence of hormones in saliva, their saliva/plasma relationship and the potential usefulness of assays of their salivary concentration.

2. Saliva: reflection of the body 

Daniela Pereira Lima, Diego Garcia Diniz, Suzely Adas Saliba Moimaz, Dóris Hissako Sumida,  Ana Cláudia Okamoto International Journal of infectious diseases. March 2010, volume 14 issue 3. Pages 184-188

Abstract

Saliva has become an important resource for evaluating physiological and pathological conditions in humans. The use of saliva has many advantages, including the simple and non-invasive method of collection and its easy, low-cost storage. With the addition of modern techniques and chemical instrumentation equipment, there has been an increase in its use for laboratory investigations, applicable for basic and clinical analyses in the fields of medicine and dentistry. The value of these methods for the diagnosis of oral and systemic diseases has been the subject of study by several researchers with the aim of increasing its use alongside complementary exams.

Hormonal analysis

The knowledge that there are steroid hormones in saliva that they can be measured has been around for more than 30 years. However, only recently has the technology reached this information, making it possible to exactly determine hormonal levels in saliva. Evaluations can be accomplished in blood samples, but this has its limitations. Most blood hormones (approximately 95%) are limited to specific proteins that carry them in the blood stream but this is only a fraction of the hormones in storage. The other 5% represent free hormones, available to move easily to their target organs and perform their functions. Saliva contains some free hormones that can be easily measured to give an exact view of those readily available in human tissue. The detection of some of these free hormones is important because their variation in saliva can be indicative of cancer progress or the possibility of a disease like Cushing’s syndrome, a disease that results from the continuous hypersecretion of endogenous cortisol, which leads to proximal musculature weakness, osteoporosis, spontaneous ecchymosis and hypocalcemia. Estrogens also can be verified in saliva and the prediction of premature birth can be detected through salivary estradiol measurement, a test approved by the FDA (Food and Drug Administration). An increase of this hormone is also an indication that a woman is in her fertile period and creating a self-test with a commercially distributed device gives her the capacity to monitor her fertile cycle.⁵⁰ Additionally, it is known that hormonal changes can lead to systemic disorders such as, for example, the diabetes mellitus.^{51, 52, 53–}

3. Salivary, but not serum or urinary levels of progesterone are elevated after topical application of progesterone one cream to pre- and postmenopausal women. O’Leary P, et al. Clin Endo (2005) 53: 615-620.

Abstract

Objective: The use of topically applied micronised (‘natural’) progesterone as a substitute for synthetic oestrogens and progestogen preparations is controversial. The aim of this study was to examine the changes in blood and salivary concentrations of progesterone following a single topical application of a progesterone cream.

Patients and Measurements: We investigated six premenopausal women in the luteal phase and six postmenopausal women to determine the short-term changes in serum, urinary and salivary progesterone concentrations following a single 64 mg topical application of micronised progesterone.

Results: Serum progesterone concentrations did not increase during the first 3 hours after application of progesterone cream, however, salivary values rose significantly in both premenopausal and postmenopausal women, consistent with the view that progesterone is absorbed and transported through the body. Salivary progesterone concentrations were significantly elevated above basal levels by 30–60 minutes and reached peak levels at 1–4 h, with mean levels approximately fivefold higher in premenopausal, than in menopausal women.

Conclusions: Salivary progesterone measurements confirm that topically applied progesterone is absorbed, despite the lack of change in serum progesterone concentrations. However, at the dose administered, serum progesterone levels do not reach those observed after oral or vaginally delivered progesterone preparations. Higher doses may be required to induce biological responses within the endometrium

4. Direct assay for progesterone in saliva: comparison with a direct serum assay. Webley GE, Edwards R. Ann Clin Biochem (1985) 22:579-585.

Abstract

Direct assays have been described for the estimation of progesterone in serum and saliva. Both methods showed good inter-assay precision over a year, and the serum assay is now being used routinely in clinical laboratories. A highly significant correlation was obtained for progesterone concentrations in matched serum and saliva samples over a wide range of concentrations. The concentrations of progesterone in daily samples taken through the menstrual cycle fell within the range quoted in previous studies. The benefits of direct saliva measurement are described with reference to the long-term assessment of irregular cycles. Care in sample collection is necessary since contamination with agents such as oral cosmetics may result in overestimation of progesterone concentrations.

5. Morning salivary cortisol versus short Synacthen test as a test of adrenal suppression Rajan S Patel, Steve R Shaw, Halena E McIntyre, Gerald W McGarry and A Michael Wallace Source Department of Otolaryngology, North Glasgow Hospitals University, NHS Trust, Glasgow, UK

Background: The short Synacthen test (SST) is the most commonly used test for the assessment of adrenal suppression. We investigated the potential of a simpler and more cost-effective procedure [morning salivary cortisol (MSC)] as an outpatient screening tool to detect adrenal suppression in patients using topical intranasal corticosteroids for rhinosinusitis.

Method: Forty-eight patients who were using topical corticosteroids underwent adrenal function assessment by way of SST and MSC measurement.

Results: Sixteen of the 48 patients had impaired MSCs. Of these 16 patients, 15 had an impaired SST (sensitivity 100%) and one had a normal SST. All patients with normal MSCs also had normal SSTs (specificity 97%).

Conclusion: The morning salivary cortisol measurement is a useful screening tool for adrenal suppression in this setting

6. The Utility and Dynamics of Salivary Sex Hormone Measurements in the National Social Life, Health, and Aging Project, Wave 2 Michael J. Kozloski and Martha K. McClintock² Journal of Gerontology

 Abstract

Objectives: Sex hormones affect physical, mental, and social health, yet their role in mediating social effects on aging is understudied. To facilitate such analyses with the National Social Life, Health & Aging Project Wave 2, we summarize the conceptual background, collection protocols, laboratory assays, and data analysis strategies for biologically active (free) levels of testosterone, estradiol, progesterone, and dehydroepiandrosterone (DHEA).

Method: Saliva from passive drool was collected from returning Wave 1 respondents and non-respondents as well as their partners. Specimens were frozen and sent to Dresden LabService GmbH for duplicate assays of biologically active steroids using identical assay kits from National Social Life, Health, and Aging Project (NSHAP) Wave 1 (SaliCap, Catalog No. RE69995). Overall, 2,772 testosterone, 2,504 estradiol, 2,714 progesterone, and 2,800 DHEA measurements are publically available for Wave 2 analyses. Through a series of weighted linear regressions, all 4 steroids are compared by gender and age and to Wave 1 measurements.

Results: Men had higher levels of both free testosterone and progesterone than women; women and men had the same levels of estradiol and DHEA. Both free testosterone and DHEA decreased with age. We also found significant wave effects for all 4 sex hormones.

Conclusion: NSHAP Waves 1 and 2 are the first U.S. probability sample studies to measure these 4 salivary sex hormones simultaneously, providing individual profiles 5 years apart. Wave 2 data demonstrate differences by gender and trends by age that are similar to those found in other saliva-based and serum-based studies of free steroid levels. The differences between waves arising from the change in assay laboratory need to be adjusted in future longitudinal analyses using NSHAP Wave 1 and Wave 2 steroid data.

7. Salivary Cortisol Levels as a Biological Marker of Stress Reaction Djordje Bozovic, Maja Racic, Nedeljka Ivkovic. Med Arh. 2013; 67(5): 374-377

Abstract
Aim: To determine the validity and psychobiological significance of salivary cortisol as a biomarker of stress in the experiments.

Results: Stress is defined as a state in which homeostasis is jeopardized by the action of various external and internal stressors. The effect of cortisol is made through specific receptors located in the cytoplasm of the target cells. Determining blood cortisol levels, which has been the most widely used method, is characterized by certain shortcomings. The process of taking blood samples from the vein is accompanied by additional stress, which results in falsely positive results. Another flaw is found in the fact that cortisol taken and measured from serum or plasma represents total cortisol, not the free, biologically active one. Cortisol response lags behind ACTH by 5-20 minutes, with peak blood levels achieved in 10-30 min. The transfer of cortisol from blood to saliva takes place rather quickly, within no more than 2-3 min.

Conclusion: Although, the studies on correlation between saliva cortisol concentrations and free levels of this hormone in blood samples are lacking, salivary cortisol offer a novel approach in research of stress biomarkers with its ease of collection and potentially wide scope for application.

8. An update: salivary hormones and physical exercise. Gatti R¹, De Palo EF. Scand J Med Sci Sports. 2011 Apr;21(2):157-69

Abstract

Saliva contains cells and compounds, of local and non-local oral origin, namely inorganic, organic non-protein, protein/polypeptide, and lipid molecules. Moreover, some hormones, commonly assayed in plasma, such as steroids, are detectable in oral fluid and peptide/protein, and non-steroid hormones have been investigated. The sports practice environment and athletes’ availability, together with hormone molecule characteristics in saliva and physical exercise behavior effects, confirm this body fluid as an alternative to serum. This review focuses on the relation between salivary steroids and psycho-physiological stress and underlines how the measurement of salivary cortisol provides an approach of self-report psychological indicator and anxiety change in relation to exercise performance. The correlation between salivary and plasma steroid hormone (cortisol, testosterone, and dehydroepiandrosterone (DHEA)) levels, observed during exercise, has been considered, underlining how the type, duration, and intensity of the exercise influence the salivary steroid concentrations in the same way as serum-level variations. Training conditions have been considered in relation to the salivary hormonal response. This review focuses on studies related to salivary hormone measurements, mainly steroids, in physical exercise. Saliva use in physical disciplines, as a real alternative to serum, could be a future perspective.

9. Current Status of Salivary Hormone Analysis Michael Gröschl.  Clinical Chemistry November 2008 vol. 54 no. 11 1759-1769

Abstract

Background: Saliva, which offers a noninvasive and stress-free alternative to plasma and serum, is a widely accepted sample source for analysis of steroids and also of certain amines and peptides. In recent years, numerous publications have described the use of salivary hormone analysis in many fields of clinical and basic research.

Content: This review provides an overview of the current applications of salivary hormone analysis. A description of the different modes of hormone entry into saliva is followed by a detailed description of analytical methods and approaches for reliable collection of saliva, including several interesting applications in diverse fields including psychiatry, stress research, clinical endocrinology, sports medicine, and veterinary medicine.

Summary: Although saliva has not yet become a mainstream sample source for hormone analysis, it has proven to be reliable and, in some cases, even superior to other body fluids. Nevertheless much effort will be required for this approach to receive acceptance over the long term, especially by clinicians. Such effort includes the development of specific and standardized analytical tools, the establishment of defined reference intervals, and implementation of round-robin trials. One major problem, the lack of compliance sometimes seen in outpatient saliva donors, requires strict standardization of both collection and analysis methods to achieve better comparability and assessment of published salivary hormone data.

The use of saliva as a sample source for hormone analysis has become increasingly attractive for clinicians and researchers because collection of saliva offers a noninvasive and stress-free alternative to collection of plasma and serum, the traditional matrices for the determination of endocrine parameters such as steroids, amines, and peptides.

For 40 years, endocrinologists have used saliva as a supplementary sample matrix. Prior reviews, from Riad-Fahmy et al. in 1982 (1) to Lewis in 2006(2), have focused on salivary analyisis of many steroids, although numerous reported studies have demonstrated that saliva monitoring is a useful alternative method for analyzing hormones of other biochemical origins.

This review provides an overview of well-established procedures and promising future methods of salivary hormone analysis and the application of these methods in fields as diverse as psychology, clinical endocrinology, fertility, sports medicine, and behavioral research.

10. Association of Salivary Levels of Cortisol and Dehydroepiandrosterone With Periodontitis in Older Japanese Adults

Aiko Ishisaka,^* Toshihiro Ansai,^* Inho Soh,^* Kiyotoshi Inenaga,^† Akihiro Yoshida,^* Chieko Shigeyama,^* Shuji Awano,^* TomokoHamasaki,^* Kazuo Sonoki,^‡ Yutaka Takata,^‡ and Tadamichi Takehara^*      Journal of Periodontology September 2007, Vol. 78, No. 9, Pages 1767-1773

Background: The relationship between periodontitis and psychoneuroimmunologic variables, such as stress-related hormones, is poorly understood. The purpose of this cross-sectional study was to investigate the associations between two kinds of stress-related hormones, cortisol and dehydroepiandrosterone (DHEA), and periodontitis in healthy community-dwelling elderly subjects aged ≥60 years.

Methods: A total of 171 subjects (85 males and 86 females) participated in this study. The subjects were independently living elderly people with a mean age of 68.4 (± 4.46) years. Stimulated whole saliva samples were collected, and hormone levels were determined. A medical questionnaire regarding medical conditions, lifestyle, and psychosocial stress also was administered. The clinical examinations included probing depth (PD), bleeding on probing, and clinical attachment level (CAL).

Results: There was a positive correlation between hormone levels and PD and CAL values. When the subjects were divided into two groups based on periodontitis severity, hormone levels were significantly higher in subjects with severe PD or CAL. Multiple regression analysis showed that higher cortisol and DHEA levels were associated significantly with greater numbers of teeth with severe PD or CAL, after adjusting for confounding variables.

Conclusions: These results suggested close relationships between the extent and severity of periodontitis and salivary levels of cortisol and DHEA in healthy elderly subjects. To the best of our knowledge, this is the first report on the association between the levels of DHEA and extensive periodontitis.

11. Human Saliva as a Diagnostic Specimen Lindsay F. Hofman² 2001 The American Society for Nutritional Sciences

Abstract

Human saliva can be easily obtained by noninvasive techniques and contains many analytes of interest for screening, diagnosis and monitoring. These include steroid and other nonpeptide hormones, therapeutic drugs, drugs of abuse and antibodies. Numerous studies in the past 40 y have shown correlations between serum and saliva levels. Both diurnal and monthly profiles of hormone levels parallel traditional serum patterns. Multiple specimens for steroid hormone analysis can be easily collected by the patient, monitor fertility cycles, menopausal fluctuations, stress and other diurnal variations. Drug doses can be monitored without inconvenient and costly visits to blood-drawing facilities. Antibody levels can be determined to screen for infectious diseases. Saliva can be collected directly by spitting into a tube or with one of several devices, each of which has its own special advantages and disadvantages. Salivary levels of steroid hormones and other analytes that are protein bound in serum reflect the unbound and active concentration of the hormone. Saliva can be used as a diagnostic specimen not only to obtain information more inexpensively and efficiently than serum, but also to provide information not readily available from serum testing.

12. Research Paper
Relationship between salivary and serum testosterone levels in response to different exercise intensities

Amy R. Lane, Anthony C. Hackney Endo crine Section – Applied Physiology Laboratory, Department of Exercise & Sport Science,²Department of Nutrition, Gillings School of Global Public Health, University of North Carolina, USA

Abstract

The anabolic hormone testosterone (T) continues to be researched extensively in exercise studies, with blood serum or plasma samples collected routinely. This method is considered both valid and reliable; however, the collection process can be invasive and difficult during exercise. For this reason, non-invasive sampling, such as saliva collections, is increasing in popularity. PURPOSE: To investigate the association between salivary and serum T before and after various intensities of exercise to assess the validity of saliva collection for T measurement. METHODS: 12 endurance-trained males (Mean±SD; VO_2max 58.2±6.4 ml/kg/min, 22±4.6 years, BMI 21.6±1.9) completed three 30-minute exercise (cycling) sessions at 40% (Low), 60% (Moderate), and 80% (High) of VO_2max as well as a 30-minute resting Control session on four separate days. Salivary (s) and serum (S) samples were assessed for T prior to (Pre), immediately post (Post), and 30 minutes post (30 Min Post) each session. RESULTS: Low exercise caused a significant (p<0.05) increase in ST, but not sT, while Moderate and High exercise caused an increase (p<0.01) in both ST and sT. A strong positive correlation (Spearman [r_s]) existed between sT and ST in the Moderate (r_s = 0.912 [p<0.001; n=36; 12 subjects x 3 measurement times-samples]), and High (r_s = 0.898 [p<0.001]) sessions, but not as strong for the Low (r_s = 0.517 [p=0.001]) session. CONCLUSIONS: These results suggest that sT and ST levels are strongly associated to exercise. However, the exercise-related sT and ST responses-changes were more closely aligned at Moderate and High intensities, suggesting a greater validity between the saliva and serum responses to higher intensity exercise.

Quantification of the Salivary Steroid Hormones Considered as Bio-markers in Clinical Research Studies and Sports Medicine

Plenis, Alina; Baczek, Tomasz

Source: Current Pharmaceutical Analysis, Volume 6, Number 3, August 2010, pp. 182-197(16)

Abstract:

Steroid hormones are important in controlling human body functions as a part of the endocrine system together with neuronal systems and the immune system. Application of the assay of the steroid hormones treated as biomarkers was recently illustrated in certain cases, for example in clinical diagnosis of stress, the Cushing syndrome, congenital adrenal hyperplasia, and infertility, as well as in the field of sports medicine. The assessment of the steroid hormones in the body fluids has so far been typically based on serum and urine. However, the use of saliva as the diagnostic medium has recently grown in popularity among the scientists and clinicians because of sample collection, which is quick, uncomplicated, and non-invasive. Moreover, steroid hormones are not bound to protein in saliva. Therefore, salivary determination is an excellent approach for evaluation of free steroid hormones.

The present study provides an overview of the analytical methods applied for salivary steroid measurements in the current clinical laboratory practice. It describes and thoroughly discusses the recent achievements associated with optimisation of the analytical conditions for the steroid assay, obtained through application of the modern separation techniques such as liquid chromatography, mass spectrometry, versus non-separation techniques such as the immunological methods. Moreover, the issues associated with optimization of the extraction procedures are presented, since sample pre-treatment is the most limiting and crucial step in analyses of biological fluids. In addition, the study evaluates the consequences of any pre-analytical variation preceding the application of the assay methodologies, stemming from the collection strategy and the subsequent storage conditions. It further provides several examples of application in diverse fields of interest such as psychology, pharmacology, clinical endocrinology, or sports medicine.

The genetics of obesity: FTO leads the way

Katherine A. Fawcett¹ and Inês Barroso Trends Genet. 2010 Jun; 26(6): 266–274

Abstract

In 2007, an association of single nucleotide polymorphisms (SNPs) in the fat mass and obesity-associated (FTO) gene region with body mass index (BMI) and risk of obesity was identified in multiple populations, making FTO the first locus unequivocally associated with adiposity. At the time, FTO was a gene of unknown function and it was not known whether these SNPs exerted their effect on adiposity by affectingFTO or neighboring genes. Therefore, this breakthrough association inspired a wealth of in silico, in vitro, and in vivo analyses in model organisms and humans to improve knowledge of FTO function. These studies suggested that FTO plays a role in controlling feeding behavior and energy expenditure. Here, we review the approaches taken that provide a blueprint for the study of other obesity-associated genes in the hope that this strategy will result in increased understanding of the biological mechanisms underlying body weight regulation.

RESEARCH

Physiogenomic analysis of weight loss induced by dietary carbohydrate restriction

Gualberto Ruaño, Andreas Windemuth, Mohan Kocherla, Theodore Holford, 

Maria Luz Fernandez, Cassandra E Forsythe, Richard J Wood,William J Kraemer and 

Jeff S Volek Nutrition & Metabolism20063:20

Abstract

Background: Diets that restrict carbohydrate (CHO) have proven to be a successful dietary treatment of obesity for many people, but the degree of weight loss varies across individuals. The extent to which genetic factors associate with the magnitude of weight loss induced by CHO restriction is unknown. We examined associations among polymorphisms in candidate genes and weight loss in order to understand the physiological factors influencing body weight responses to CHO restriction.

Methods: We screened for genetic associations with weight loss in 86 healthy adults who were instructed to restrict CHO to a level that induced a small level of ketosis (CHO ~10% of total energy). A total of 27 single nucleotide polymorphisms (SNPs) were selected from 15 candidate genes involved in fat digestion/metabolism, intracellular glucose metabolism, lipoprotein remodeling, and appetite regulation. Multiple linear regression was used to rank the SNPs according to probability of association, and the most significant associations were analyzed in greater detail.

Results: Mean weight loss was 6.4 kg. SNPs in the gastric lipase (LIPF), hepatic glycogen synthase (GYS2), cholesteryl ester transfer protein (CETP) and galanin (GAL) genes were significantly associated with weight loss.

Conclusion: A strong association between weight loss induced by dietary CHO restriction and variability in genes regulating fat digestion, hepatic glucose metabolism, intravascular lipoprotein remodeling, and appetite were detected. These discoveries could provide clues to important physiologic adaptations underlying the body mass response to CHO restriction.