Integration of salivary biomarkers into developmental and behaviorally-oriented research: problems and solutions for collecting specimens.

Saliva has been championed as a diagnostic fluid of the future. Much of the attention that saliva receives as a biological specimen is due to the perception that the nature of sample collection is quick, uncomplicated, and non-invasive. In most cases, this perception matches reality; however, in some special circumstances and populations collecting saliva can be unexpectedly difficult, time consuming, and may not yield sufficient sample volume for assay. In this report, we review the nature and circumstances surrounding some of these problems in the context of developmental science and then present alternatives that can be used by investigators to improve the next generation of studies. We expect our findings will ease the burden on research participants and assistants, reduce the rate of missing values in salivary data sets, and increase the probability that salivary biomarkers will continue to be successfully integrated into developmental and behaviorally-oriented research.

Oxytocin is not a valid biomarker when measured in saliva by immunoassay.

The integration of oxytocin (OT) into behavioral science seems to hold considerable promise for advancing our understanding of human health and development but methodological issues restrict the measurement of OT in large studies, in everyday social settings, or when repeated sampling is required. Measuring OT in saliva could overcome many of these limitations. In this paper, we rigorously evaluate the feasibility of doing so. A series of experiments leads to the conclusion that saliva does not contain oxytocin in measurable amounts, and that OT is not a valid salivary biomarker when measured by currently available immunological methods. Levels of immuno-reactive OT in saliva are primarily due to non-specific interference with antibody-antigen binding. We can state with a high degree of certainty that measurement of OT in saliva does not yield meaningful indices of individual differences or intra-individual change.

The "trouble" with salivary testosterone.

In a series of studies, we identify several specific issues that can limit the value of integrating salivary testosterone in biosocial research. 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. There are 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. The findings have important implications at several levels of analysis for research that aims to test biosocial models of testosterone--behavior relationships. Recommendations are provided to steer investigators around these "troubles" with salivary testosterone.

Blood contamination and the measurement of salivary progesterone and estradiol.

The impact of blood leakage due to microinjury to the oral cavity on the measurement of salivary reproductive hormones was examined. Saliva samples were collected before, immediately after, and then every 15 min for 1 h following vigorous tooth brushing. Blood in saliva was quantified by visual inspection of discoloration and an immunoassay for transferrin. Levels of progesterone increased, and levels of estradiol decreased, in saliva after microinjury. These changes were present only immediately after microinjury. The findings have implications for the use of salivary assays in biobehavioral research, short-term dynamic investigations, pharmacokinetic analyses, and studies of chronobiological changes in progesterone and estradiol levels.

Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva.

The impact of blood leakage due to microinjury to the oral cavity on the measurement of salivary hormones was examined. Saliva samples were collected before, immediately after, and then every 15 min for 1 h following vigorous tooth brushing. Blood in saliva was quantified by visual inspection of discoloration, Hemastix reagent strips to detect hemoglobin, and an immunoassay for transferrin. The presence of blood in saliva immediately after microinjury was confirmed by all methods. Hemoglobin and transferrin levels remained elevated over baseline for at least 30 min. Levels of salivary testosterone increased over baseline and remained elevated for 30 min in response to microinjury. Microinjury induced change in salivary testosterone was more closely associated with the change in transferrin than hemoglobin levels or discoloration ratings. On average, levels of salivary dehydroepiandrosterone (DHEA) did not increase in response to microinjury. However, individual differences in microinjury induced change in DHEA were associated with discoloration ratings. Salivary cortisol levels, on average, were neither responsive to microinjury, nor were individual differences in cortisol change associated with blood contamination measures. Neither diurnal nor gender-related differences in baseline hormone levels predicted the impact of blood leakage on quantitative salivary measurements. The findings suggest ecologically valid minor-to-moderate level microinjuries to the oral cavity have negligible effects on the measurement of salivary cortisol, but may be important to quantify and control when assessing other hormones especially testosterone.