Validation of salivary testosterone as a screening test ...

The Aging Male, September 2006; 9(3): 165–169

Validation of salivary testosterone as a screening test for male hypogonadism

JOHN E. MORLEY1,2, H. MITCHELL PERRY III1,2, PING PATRICK1, CHARLES M. DOLLBAUM3,4, & JOHN M. KELLS3 Division of Geriatric Medicine, Saint Louis University, St. Louis, Missouri 63104, 2GRECC, St. Louis VAMC, St. Louis, Missouri 63125, 3Aeron LifeCycles Laboratory, San Leandro, California 94577, and 4University of California, San Francisco, California 94149, USA

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Abstract Background. Saliva collection is an easy, non-invasive method to measure hormones. Methods. Two studies were performed. In the first, a convenience sample of 1454 males who had submitted saliva for salivary testosterone measurements were studied. In the second study, we intensively studied symptoms and measurements of total testosterone, free testosterone symptoms and measurements of total testosterone, free testosterone and bioavailable testosterone in relationship to salivary testosterone in 127 men. A secondary endpoint was to examine the relationship of salivary testosterone to hypogonadal symptoms in the ADAM and AMS questionnaires. Results. In the first study, we have shown that salivary testosterone, measured in 1454 males aged 20 to 89 years, declines by 47% over the lifespan. In the second study, salivary testosterone was strongly correlated with bioavailable testosterone (p 5 0.000001) calculated free testosterone (p 5 0.00001) and total testosterone (p 5 0.002). Salivary testosterone was significantly related to hypogonadal symptoms on the St. Louis University ADAM questionnaire and the Aging Male Survey. Conclusions. These studies support the use of salivary testosterone as an acceptable assay for screening for hypogonadism. Salivary testosterone is not a better assay than other measures to diagnose hypogonadism.

Keywords: Saliva, bioavailable testosterone, andropause, aging male survey, ADAM Abbreviations: ST, salivary testosterone; ADAM, Androgen Deficiency in Aging Male; AMS, Aging Male Survey; SHBG, sex hormone binding globulin; BT, bioavailable testosterone; TT, total testosterone; CFT, calculated free testosterone

Introduction Saliva collection is an easy, non-invasive method to measure hormones. It is believed that for many hormones salivary concentrations represent the concentration of non-protein bound forms in the blood [1,2]. Most steroid hormones can be appropriately measured in the saliva, but the measurement of thyroxine has been problematic [3,4,5,6,7,8,9]. Landman et al. [10] demonstrated testosterone in the saliva in 1976 and this has been confirmed by multiple other studies [11,12,13,14,15,16]. In 1980, Gaskell et al. [17] reported the presence of testosterone in saliva using gas chromatography – mass spectroscopy and reported a range of salivary testosterone of 200 to 500 pmol/L for normal men. It is recognized that the salivary glands are target organs for testosterone and, thus, modification of testosterone by the salivary glands is a possibility [18]. It is now well accepted that testosterone levels decline over the lifespan [19,20,21]. It is believed that this decline is associated with a constellation of

symptoms that has been termed the andropause [22,23,24]. Recently data has emerged to suggest that the frailty syndrome [25,26] and loss of muscle mass (sarcopenia) [27,28] may be associated with this agerelated testosterone decline [29,30,31,32,33]. Two questionnaires examining the symptoms of hypogonadism have recently been developed viz. the ADAM questionnaire and the Aging Male Survey [34,35]. In this study we have examined the effect of age on salivary testosterone in a large group of men. Then in a smaller group of men we have validated the salivary testosterone levels against circulating bioavailable (free and albumin bound) testosterone and the free testosterone index. Finally, we examined the relationship of salivary testosterone to symptoms of hypogonadism. We hypothesized that salivary testosterone may be a suitable screening test for males with symptomatic hypogonadism. In the first study, the primary endpoint was the relationship of salivary testosterone to age. The primary endpoints in the second study were the relationship to total, free and bioavailable testosterone. The secondary endpoints

Correspondence: John E. Morley, Division of Geriatric Medicine, Saint Louis University School of Medicine, 1402 S. Grand Blvd., M238, St. Louis, Missouri 63104, USA. Fax (314) 977-8409. E-mail: [email protected] ISSN 1368-5538 print/ISSN 1473-0790 online Ó 2006 Informa UK Ltd. DOI: 10.1080/13685530600907993

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were the relationship of salivary testosterone to symptoms of hypogonadism as measured by the ADAM and AMS.


Methods Bioavailable (BT) and total testosterone (TT) were measured as previously described in detail by us [36,37]. The inter-assay and intra-assay sensitivity for BT was 10.4% and 5.8% respectively and for total testosterone 6.7% and 7.7%. Sex hormone binding globulin was measured utilizing radioimmunoassay utilizing a commercially available kit (Endocrine Sciences, Calabassas Hill, CA). The interand intra-assay coefficients of variation were 6.7% and 8.2%. The calculated free testosterone index (cFT) was calculated as described by Vermeulen et al. [38]. Salivary samples were collected early in the morning prior to brushing, eating or flossing, simultaneously with blood draws. All salivary samples were collected directly into 10 ml polypropylene tubes. Sugarless gum (Eclipse, Wrigley, IL) was supplied with the collection tube and it was suggested that participants chew the sugarless gum while collecting saliva to stimulate saliva flow. Use of cotton-based sampling was avoided as it has previously been shown to interfere with salivary immunoassay results. Samples were frozen after collection and stored at 7708C prior to shipping to the Aeron laboratory on dry ice for testing. Upon receipt samples were held at 7708C to testing. For testing, samples were thawed and centrifuged at 1500 g for 15 minutes to sediment particulate and viscous material. Aliquots of the supernatants were taken for testing. The salivary testosterone assay (Aeron Salivary Bioavailable Testosterone-SBA-T) is a modification of the Diagnostic Systems Laboratories, Inc. (Webster, TX) 125I double antibody test kit for the quantification of total testosterone in serum. Kit standards are diluted to give final concentrations of 0, 5, 10, 20, 50, 100, 200 500, 750 pg/ml. Internal kit controls I and II are diluted to give values of 20 and 200 pg/ml respectively. All samples are run in duplicate. To 250 microlitres of standards, controls and samples – each containing 10% charcoal stripped fetal calf serum –50 microlitres of primary antibody solution and 250 microlitres of I125 tracer solution are added and the tubes were vortexed and incubated for 70 min at 378C. After incubation, 500 microlitres of precipitating agent was added to each tube. The tubes are vortexed and incubated for 20 minutes on ice and then centrifuged at 1500 g for 30 minutes. Supernatant is then decanted and tubes are counted in a Genesis 5000 multiwell gamma counter. Results are calculated from log-linear regression. Intra-assay variability was 5.4% for low, 2.8% for middle range and 2.4% for high range. The respective inter-assay variability was 9.4%, 3.0%, and 3.3%.

The St. Louis University ADAM Questionnaire [34] and the Aging Male Survey (AMS) [33] were administered at the same time as blood and saliva was collected. For the first study 1454 subjects were used for the age comparison of salivary testosterone levels in males. For the validation study, saliva and blood was collected from 127 men age 23 to 80 years (mean 53.1+1.12 years). This study was approved by the St. Louis University Institutional Review Board. For the first study, 1454 subjects were used for the age comparison of salivary testosterone levels in males. These subjects had submitted their saliva sample to be measured in a commercial laboratory. In each case they had given their age as a part of the laboratory’s requirements. The values obtained were analysed in comparison to their age. No other data was available for this group. For the validation study, saliva and blood was collected from a convenience sample of 127 men aged 23 to 80 years (mean 53.1+1.12 years). Subjects were provided the results of the tests. The two groups of subjects did not overlap. This study was approved by the St. Louis University Institutional Review Board and informed consent was obtained. Statistical analyses were performed using a personal computer and a commercially available statistical software package (Statistica, Statsoft, Oklahoma City, OK). Statistical comparisons used analysis of variance, student’s t test and regression analysis. Results Figure 1 demonstrates the fall in ST in 1454 males aged 20 to 89 years of age. ST declined in an age dependent fashion with a drop in mean values of 47% from the second to the eighth decade of life (p 5 0.001). The results by decade are provided in the figure. No data is available on this cohort to provide further analysis. Figure 2 shows that ST in the second cohort was correlated with BT (r ¼ 0.66412, p 5 0.000001), CFT (r ¼ 0.4211, p 5 0.00001) and TT (r ¼ 0.3236, p 5 0.002).

Figure 1. Effect of age on salivary testosterone levels in males.

Salivary testosterone screening test for male hypogonadism In the second study, the secondary endpoints were the ADAM and AMS score. All subjects completed both questionnaires. ST was significantly related to a positive ADAM score (p 5 0.001) and a total AMS score (p 5 0.0001). On the ADAM questionnaire ST was positively correlated with decreased libido, lack of energy, decreased strength and endurance, height loss, decreased enjoyment of life, erections being less strong, falling asleep after dinner and deterioration in work performance (Table I). On the AMS questionnaire ST was positively correlated with sleep problems, physical exhaustion/lacking vitality, decrease in muscle strength, having hit rock bottom, decrease in ability to perform sexually, decrease in

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morning erections and decrease in sexual desire (Table II). Discussion This study demonstrates that ST is correlated with TT, CFT and BT. This adds to the previous literature demonstrating that ST correlates with free testosterone by dialysis [39]. There is a circadian rhythm of ST similar to that observed in serum measures of testosterone [40]. A three day stimulation test with human chononic gonadotrophin increased salivary testosterone levels [41]. Males with documented hypogonadotrophic hypogonadism

Table I. Comparison of salivary testosterone with symptoms on the St. Louis ADAM questionnaire.


Salivary testosterone (pg/ml) ADAM questions 1. Decreased libido 2. Lack of energy 3. Decreased strength and/or endurance 4. Loss of height 5. Decreased ‘enjoyment of life’ 6. Sad and/or grumpy 7. Erections less strong 8. Deterioration in ability to play sports 9. Falling asleep after dinner 10. Deterioration in work performance

Yes

No

p

8.2 + .4 8.0 + .4 8.1 + .4

10.1 + .8 9.7 + .6 10.0 + .7

0.01 0.05 0.05

8.0 + .7 8.6 + .6

9.2 + .4 9.1 + .5

NS NS

8.9 + .7 8.4 + .4 8.3 + .5

8.9 + .4 9.7 + .6 9.3 + .5

NS 0.05 NS

7.0 + .5 8.4 + .6

9.4 + .5 9.0 + .4

0.01 NS

(Significant values are underlined); NS ¼ not ssignificant.

Table II. Comparison of salivary testosterone with symptoms from the Aging Male Survey. Salivary testosterone Questions Decline in well-being Joint pain/muscular ache Excessive sweating Sleep problems Increased need for sleep/often tired Irritability Nervousness Anxiety Physical exhaustion/lacking vitality Decrease in muscular strength Depressive mood Passed your peak Burnt out/rock bottom Decrease in ability/frequency to perform sexually Decrease in number of morning erections Decrease in sexual desire/libido Figure 2. Correlation of salivary testosterone with bioavailable testosterone, calculated free testosterone and total testosterone.

(NS ¼ not significant).

r

p

70.0011 70.0229 70.0729 70.2404 70.0976 70.1594 0.1120 0.1627 70.2333 70.1809 70.0523 70.2691 70.2250 70.909

NS NS NS 0.01 NS NS NS NS 0.01 0.01 NS 0.01 0.01 NS

70.2872

0.01

70.1371

NS


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have lower ST levels than age-matched controls [42]. Testosterone administration increased ST in parallel with serum TT [43]. Overall, this data supports the legitimacy of ST as an assay to measure bioeffective (tissue available) testosterone. Further validation for the use of ST comes from its high correlation with hypogonadal symptoms. The ADAM questionnaire has been previously validated as having excellent sensitivity [34,44,45]. It has poorer specificity and, in particular, persons with depression are very likely to answer positively on this questionnaire. Preliminary data suggests that the AMS performs similarly to the ADAM questionnaire. The correlations of these two questionnaires with ST further support the concept that these questionnaires are measuring symptoms of hypogonadism in men across the lifespan. The combination of ST both correlating with BT and symptoms makes it an acceptable measure of hypogonadism. It should be recognized that some other co-variates, e.g. age, may be responsible for the concomitant decrease in testosterone and the symptoms reported in the questionnaire. Five longitudinal studies and numerous crosssectional studies have demonstrated that testosterone declines with aging while SHBG increases [19,20,21,46,47]. Previously, some small studies have demonstrated a decline in ST with aging [48,49,50]. Our large study confirms the decline in ST with aging. Dabbs [51] has previously demonstrated week to week variability in ST. This finding is of similar magnitude to that previously reported by Vermeulen et al. [52] for serum TT and by us [53] for BT. Salivary flow rate changes affect the concentration of conjugated steroid metabolites, such as dehydroepiandrosterone sulfate, leading to a decrease in their salivary concentration with increased salivary flow rates [54]. However, like cortisol, testosterone levels in the saliva appear to not show a dependence on salivary flow rate [54]. A number of advantages of measuring testosterone in the saliva have been previously delineated [1]. These include (I) the avoidance of stress associated with venipuncture (II), the fact that salivary testosterone levels reflect the bioavailable (or free) plasma fraction, (III) samples can be collected at home and, (IV) multiple sample collection to study normal physiology is facilitated. It should be noted that previous wide variation in normal salivary testosterone levels appears to be due to the use of nonspecific antibodies to measure testosterone. This study together with previous studies provides strong evidence that salivary testosterone is an acceptable assay for screening for hypogonadism. In view of the poor correlation of total testosterone with bioavailable and free testosterone assays, and the difficulty in obtaining these assays, ST may be the assay of choice for clinical use in diagnosing hypogonadism.

Acknowledgements For research support the authors would like to thank Solvay Pharmaceuticals, Marietta, GA, and AeronCycles Laboratory, San Leandro, California.

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