Diagnostic significance of free salivary testosterone measurement using a direct luminescence immunoassay in healthy men and in patients with disorders of androgenic status.

The accurate measurement of testosterone remains a challenge. The determination of the blood testosterone concentrations in serum by conventional immunoassays is inaccurate in men and even more so in females and children. A new luminescence enzyme immunoassay (LIA) has been developed and validated. The high analytical (8.7 pmol/L) and functional (17.3 pmol/L) sensitivity allows the quantification of the very low concentration in saliva, as well as in serum, after 1/40 dilution. This study measured salivary testosterone levels and compared the results with the free levels calculated from total testosterone and sex hormone-binding globulin in eugonadal and hypogonadal men. Salivary testosterone concentrations in healthy men in morning hours were 369 pmol/L (mean), range 263-544 pmol/L, which was statistically significantly higher than that in men with androgen deficiency, 215 pmol/L (mean), range 51-249 pmol/L. Repetitive determination of free testosterone concentrations in saliva (once a week for 5 weeks) showed high stability of results over time, with coefficient of variation 9% (range 5-23%). In this study we showed that free salivary testosterone levels in morning samples correlated well with calculated free testosterone in blood, both in healthy men (R = 0.754, P = 0.001), and in patients with androgen deficiency (R = 0.889, P = 0.0001), though in cases with very low testosterone, salivary concentrations were systematically higher than calculated free testosterone levels in blood.

Some new aspects of 17alpha-estradiol metabolism in man.

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.