The “trouble” with salivary testosterone
Introduction
The ability to measure biological variables non-invasively in saliva has created many opportunities for behavioral scientists to test biosocial models of individual differences and intra-individual change in mood, cognition, behavior and psychopathology (Booth et al., 2000, Dabbs, 1993, Kirschbaum et al., 1992, Malamud and Tabak, 1993). As the number of studies doing so has increased, special circumstances capable of compromising the value of the information generated have been documented (e.g., Magnano et al., 1989, Schwartz et al., 1998). Over the past 10 years, our studies have documented that these issues are especially salient with respect to the measurement of testosterone in saliva. In this paper, we comprehensively review these problems and pitfalls to update this information (Dabbs, 1993) and steer the next generation of studies clear of these “troubles” with salivary testosterone.
There has been renewed interest among researchers in the relationship between testosterone and a variety of behavioral (e.g., Dabbs and Morris, 1990), physiological (e.g., Walker et al., 1980), social (e.g., Booth and Dabbs, 1993) and health constructs (e.g., Boas et al., 1996, Booth et al., 1999). Access to non-invasive means of measuring testosterone (in saliva) has in part rekindled this interest. Monitoring testosterone in saliva has several obvious advantages over doing so in serum or urine. Saliva collection procedures afford researchers the ability to conduct repeated sampling over the course of minutes, hours, days or longer (Malamud and Tabak, 1993). Research assistants, caregivers and subjects themselves with minimal training can easily collect saliva samples in most circumstances. Also the reliability, precision, accuracy and analytical recovery of immunoassays designed to measure salivary testosterone are now well documented (e.g., Granger et al., 1999). The ease of collection and recent commercial availability of salivary assay protocols have contributed to the impression that salivary testosterone is “ready” to be incorporated into biosocial research on a widespread basis (Dabbs, 1990). However, there are a number of studies reporting accounts of special circumstances with potential to significantly influence the external validity of salivary testosterone results (Dabbs, 1991, Granger et al., 1999). A more fine grained examination reveals wide gaps in information available to guide researchers in the utility of this measurement approach.
In this paper, we document that salivary testosterone measurements can be substantially influenced during the process of sample collection, are susceptible to interference effects caused by the leakage of blood (plasma) into saliva and are sensitive to storage conditions when samples have been archived. We describe gender differences in salivary testosterone levels and variance, the serum–saliva association, the relationship of salivary testosterone to age and pubertal development and the stability of individual differences in salivary testosterone levels over time. Implications for testing biosocial models of testosterone–behavior relationships are discussed and guidelines and recommendations are provided.
Section snippets
Saliva sample collection
A series of experiments have extensively evaluated the effects of different sample collection techniques on the measurement of a variety of salivary biomarkers (Dabbs, 1991, Granger et al., 1999, Schwartz et al., 1998, Shirtcliff et al., 2001a). Here, we review that materials commonly used in the literature to absorb sample (i.e., cotton and polyester swabs) or stimulate saliva flow (i.e., powdered drink-mix crystals and chew gum) have the potential to substantially change salivary testosterone
Blood contamination of saliva
Ten years ago, the literature warned that when blood is present in the oral mucosa, quantitative estimates of hormones measured in saliva may be compromised (Malamud and Tabak, 1993). Blood (and its components) can leak into the oral mucosa as a result of microinjuries such as burns or abrasions. To be a meaningful index of systemic bioactivity, quantitative estimates of testosterone in saliva must be highly correlated with the levels in serum. A high serum–saliva correlation depends on
Correspondence of testosterone levels measured in saliva and serum
As noted above, to meaningfully reflect systemic bioactivity, quantitative estimates of testosterone in saliva must be highly correlated with the levels in serum. In a recent paper (Shirtcliff et al., 2002), we documented a sex difference in the association between salivary and serum testosterone measures, and then determine if this sex difference would affect the detection of testosterone–behavior associations.
Participants in this analysis were 20 male and 20 female Penn State University
Sample storage and archiving
As the collection of saliva becomes more routine, samples will more commonly be archived to be assayed in the future. In this analyses, we evaluate week-to-week change in levels of testosterone in saliva samples when stored up to four weeks at 4 °C in a regular household refrigerator. Also, the long-term stability of testosterone levels in saliva was tested every 6 months when stored up to two years at −20 °C in a frost free (i.e., freeze–thaw cycling) regular household freezer, and also at −40
Modeling individual differences in salivary testosterone
Contemporary theorists speculate about reciprocal effects between hormones and behavior, and assume such effects are heavily dependent on intrinsic individual differences as well as characteristics of the social ecology (Booth et al., 2000, Brooks-Gunn et al., 1994, Gottlieb, 1992, Susman, 1997). To test these complex ideas requires a focus on identifying sources of individual differences, and employing methodologies that gather multiple waves of hormone and behavioral data over time.
General conclusion
Contemporary theorists consider testosterone–behavior relationships to be dynamic, having reciprocal, and potentially non-linear, effects that are highly dependent on intrinsic characteristics and features of the social environment (Booth et al., 2000, Brooks-Gunn et al., 1994, Campbell et al., 1997, Susman, 1997). In order to extend our understanding of these effects to new limits, investigators have begun to take advantage of saliva sampling to measure testosterone in ecologically valid
Acknowledgements
We acknowledge Robert Sapolsky’s creativity as the title of this paper is a play on the title of his book, “The Trouble with Testosterone”. Thanks are due to Ruth Merritt, Jodi Heaton, Jessica King, Jyotika Mirchandani, Andrew Kurscher, Andrew Hackett, Erica Schreffler, and Stefanie Bachman during data collection, and Laurie O’Brien, Carla Slike, Carol Boob, and Skip Nelson for technical assistance with the assays. The preliminary findings and comments shared with us by our colleagues Vangie
References (33)
Salivary testosterone measurements: reliability across hours, days, and weeks
Physiology & Behavior
(1990)Salivary testosterone measurements: collecting, storing, and mailing saliva samples
Physiology & Behavior
(1991)- et al.
Testosterone and conduct problems
Journal of the American Academy of Child and Adolescent Psychiatry
(1994) - et al.
Salivary testosterone determination in studies of child health and development
Hormones and Behavior
(1999) - et al.
Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva
Hormones and Behavior
(2004) - et al.
Use of salivary biomarkers in biobehavioral research: cotton based sample collection methods can interfere with salivary immunoassay results
Psychoneuroendocrinology
(2001) - et al.
Gender differences in the validity of testosterone measured in saliva by immunoassay
Hormones and Behavior
(2002) - et al.
Salivary testosterone levels in male adolescents with cystic fibrosis
Pediatrics
(1996) - et al.
Testosterone and men’s marriages
Social Forces
(1993) - et al.
Testosterone and men’s health
Behavioral Medicine
(1999)
Biosocial perspectives on the family
Journal of Marriage and the Family
Testosterone and child and adolescent adjustment: the moderating role of parent–child relationships
Developmental Psychology
Studying links between hormones and negative affect: models and measures
Journal of Research on Adolescence
Aggression and testosterone: testing a bio-social model
Aggressive Behavior
Salivary testosterone measurements in behavioral studies
Annals of the New York Academy of Sciences
Testosterone and antisocial behavior in a sample of 4462 men
Psychological Science
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