Because the portion of circulating testosterone that is not bound to SHBG is generally believed to represent the biologically active fraction, many laboratory methods for determining non-SHBG-bound testosterone or free testosterone have been developed. Because SHBG levels are low with obesity or hyperinsulinemia, established risk factors for the development of coronary artery disease, such methods are essential for research seeking to link testosterone to cardiovascular endpoints. Of the methods available, there is a high positive correlation between the level of free testosterone by equilibrium dialysis, the gold-standard, non-SHBG testosterone (bioavailable testosterone), and the free-testosterone level calculated from the levels of total testosterone and SHBG (171,172).
The direct free-testosterone assay was developed as a single-step, nonextraction method (125) in which an I125-labeled testosterone analog competes with free testosterone in plasma for binding to a testosterone-specific antiserum that has been immobilized on a polypropylene assay tube. The majority of commercial laboratories measure free testosterone using analog assay kits. These assays propose to adjust the total testosterone level for the effect of a high or a low level of SHBG. However, with current direct free-testosterone assay kits, the percentage of free testosterone is unrelated to the level of SHBG in normal men. Moreover, as shown in Fig. 3, like total testosterone, free-testosterone levels in serum samples from adult men measured with analog methods correlate positively with the SHBG level (173). Age-related reference ranges for men have been reported for analog assays (174); however, the free-testosterone level determined with analog assays provides essentially the same information as the total testosterone level in men, and these assays are not, in theory, appropriate for research on the metabolic cardiovascular syndrome.
Using an analog assay, free testosterone, like total testosterone, has been inversely correlated with insulin levels (fasting and 2-h postprandial) (175). Furthermore,
Fig. 3. Relationship between sex hormone-binding globulin and free testosterone levels estimated using an analog assay from Diagnostic Systems Laboratories (Webster, TX) in normal men.
free-testosterone levels by analog methods were reduced in men with type 2 diabetes (63), as well as in men who subsequently developed diabetes (14,64). Another study found that free-, as well total, testosterone levels were reduced in men with hypertension (176). De Pergola et al. (157) found that the thrombotic factors PAI-1 antigen, fibrinogen, and factor VII were inversely associated with free-testosterone levels. Although the method for free-testosterone measurement in that study was not stated, the range of values suggests that an analog method was used. Overall, results for free testosterone must be evaluated, with attention to methodology, and the conclusions of some studies will require confirmation with more specific methods.
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