Assessment of free testosterone

The direct measurement of free testosterone in serum is based on the same principles governing the assay of free thyroid hormones and has been extensively considered and reviewed by R. Ekins in the past (Ekins 1990). As indicated above, serum testosterone exists in an equilibrium between free and protein-bound fractions, an equilibrium which is invariably disturbed by all methods of free hormone measurement, a factor that should be kept in mind when choosing a method and analyzing the data. The methods of reference for free hormone analysis are equilibrium dialysis and ultrafiltration, which should be used for research purposes and to validate other systems.

21.4.4.1 Equilibrium dialysis

In equilibrium dialysis the serum (dialysand) is put in contact with a buffer (dialysate) though a membrane which allows the passage of low molecular weight compounds (e.g. free hormones) but retains the binding proteins. As a consequence of the passage of free hormone molecules to the dialysate, new hormone molecules will dissociate from the binding proteins until a new equilibrium is reached and the free hormone concentration is the same on the two sides of the membrane. The free hormone can now be measured in the dialysate either directly (e.g. by RIA) or indirectly by knowing the total hormone concentration and assessing the percentage of added labeled hormone passed in the free fraction. Provided that the ion composition of the buffer does not interfere with the equilibrium constant (K), dialysis is thermodynamically equivalent to serum dilution and leads to a reduction of the free hormone concentration and to dissociation of new hormone from the binding proteins until a new equilibrium is established in the system. At equilibrium the original free hormone concentration is therefore only "approximately" maintained because, since the total hormone concentration is constant, the final, measured free hormone concentration will be somewhat diluted and lower than that in the original sample. This effect can be regarded as negligible if the relative free hormone concentration is low, as in the case of free thyroxin (0.02%), but might become relevant in the case of free testosterone (2%) so that the buffer volume against which the sample is dialyzed should be kept to a minimum. In this respect, it is the total volume of dialysand + dialysate which determines the dilution factor while the position of the dialysis membrane between the two compartments, i.e. the individual volume of the two compartments, is irrelevant for the free hormone concentration which, at equilibrium, will be the same on the twosides (Ekins 1990).

21.4.4.2 Ultrafiltration

The problem of sample dilution is avoided in the case of ultrafiltration of undiluted serum, the second reference method in free hormone determination. In this procedure a serum sample is centrifuged through a membrane with an appropriate molecular weight cutoff. Only free hormone and low molecular weight compounds will be collected in the ultrafiltrate at a concentration equal to that in the original sample. The free hormone can be directly assessed by RIA of the ultrafiltrate or by indirect measurement of the relative fraction of labeled hormone added to the original samples which is recovered in the ultrafiltrate (Vlahos etal. 1982). Direct measurement by RIA is preferable both in ultrafiltration and in equilibrium dialysis, since the impurities of the tracers can result in inaccurate estimation of the free fraction. Ultrafiltration devices are commercially available (e.g. Centrifree® micropartition system, Millipore).

Possibly ultrafiltration may be limited if non-filterable binding competitors are present in the sample, or if binding proteins interact with the membrane. This will result in progressive increase of the free hormone concentration in the filtrate. However, since the ultrafiltration time is rather short (one hour or less) compared to dialysis (several hours), the possible changes in the equilibrium ensuing from these and other factors may be assumed to be negligible.

21.4.4.3 Direct free testosterone RIA

These methods are based on the concept that if an antibodyisadded to a serum sample, only free hormone will bind to it and the antibody occupancy will depend on the free hormone concentration. As a result, however, protein-bound hormone will dissociate and a new equilibrium will be established. Therefore, the antibody concentration should be kept "small" enough to minimize the depletion of the protein-bound pool, i.e. not more than 1% of total hormone should be displaced from the binding proteins to the antibody. Quantification of the antibody-bound hormone, i.e. the free hormone concentration, can be achieved indirectly by knowing the total hormone concentration, adding labeled hormone and measuring the fraction of it which is taken up by the antibody ("labeled hormone antibody uptake"). Alternatively, a two-step approach involves adding the sample to a solid phase antibody, washing off the unbound serum components and adding labeled hormone which will be bound by the residual, unoccupied antibodybinding sites. Since the amount of antibody is limited, the higher the free hormone concentration, the lower the number of unoccupied antibody sites at the end of the first incubation, the lower the antibody occupancy by labeled hormone at the end of the second incubation. In order for this method to work, a two-step approach, with removal of serum after the first reaction of the antiserum with the free hormone, is necessary because if the labeled hormone interacts with the serum binding proteins, this would impair the estimation of antibody occupancy by the tracer. The two-step approach, however, is not necessary if one uses a labeled compound which is totally non-reactive with serum proteins, but can be recognized by the solid phase antibody present in a limited amount and which competes for binding with the free hormone in the sample. This is the principle of the free "analog" testosterone assay on which some popular commercially available kits are based.

The direct measurement of free testosterone in serum based on the labeled hormone "analog" is valid, provided that the analog does not interact with the serum proteins, a condition which is currently not met by commercial kits. In fact, neither the identity of the analog tracer, nor the validation of the kit (showing the absence of interactions with the serum protein) is usually disclosed by the manufacturer. On the contrary, the "analog" principle is often not even mentioned or is misrepresented in the instruction accompanying the kits, which are often validated only against other kits and not against dialysis or ultrafiltration. It should be kept in mind that, in practice, finding a hormone analog totally unreactive with serum proteins is very difficult and several studies have shown that such an interaction indeed occurs, resulting in inaccurate measurements of free testosterone. In this respect it is interesting that serum free testosterone measured by an "analog" method accounts for 0.5-0.65% of total testosterone, while equilibrium dialysis and ultrafiltration give values of 1.5-4%, revealing inconsistencies between the different approaches (Rosner 1997; Winters etal. 1998). In a direct comparison, free testosterone values measured by a bestseller "analog" kit were only 20-30% of those measured by equilibrium dialysis (Vermeulen et al. 1999). For these reasons it is recommended that, if an "analog" method is to be considered for routine free testosterone determination, in-house validation of the kit should involve comparison with dialysis or ultrafiltration and estimation of the binding of the analog tracer to endogenous proteins e.g. by adding exogenous SHBG (e.g. serum from pregnant women) and by estimating tracer binding to concanavalin A-bound SHBG after chromatography ofthe serum samples (Winters etal. 1998). Unfortunately, the kits for direct free testosterone measurement presently available do not measure what they claim to do and give inaccurate results (Rosner 2001). Their use should be discouraged.

21.4.4.4 Salivary testosterone

Salivary testosterone is considered to be a good index of serum free testosterone and a highly significant correlation with serum total and free testosterone has long been known (Wang etal. 1981). As other steroid hormones, free testosterone enters saliva though passive diffusion across the acinar cells of the salivary glands. Testosterone can be measured directly in saliva by RIA or FIA with or without extraction (Wang et al. 1981; Schurmeyer et al. 1984; Tschop et al. 1998; Granger et al. 1999). Monitoring salivary testosterone levels may be particularly useful in studies involving children or subjects poorly compliant with blood withdrawal and is currently widely used in behavioral studies (Granger et al. 1999). It can also be used to monitor the pharmacokinetics of testosterone preparations used for substitution therapy without requiring the patient to come to the laboratory frequently (Tschop et al. 1998). Saliva testing of testosterone concentration is also very popular in the internet, where several US companies advertise and sell at-home hormone test kits.

21.4.4.5 Bioavailable testosterone

Several lines of evidence suggest that not only free testosterone but also albumin-bound testosterone is available to the target tissues for biological activity (Manni etal. 1985). Therefore, the non SHBG-bound testosterone is called "the bioavailable testosterone". This parameter can be measured based on the property of ammonium sulfate to precipitate SHBG together with the steroids bound to it. Tracer amounts of labeled testosterone are added to the samples and, after allowing for equilibration with endogenous testosterone, an equal volume of a saturated solution of ammonium sulfate is added (final concentration 50%) and SHBG is separated by centrifugation. The percentage of labeled testosterone remaining in the supernatant represents an estimation of bioavailable testosterone, which can be calculated knowing the total testosterone concentration of the sample (Manni etal. 1985). Alternatively, testosterone can be measured directly in the supernatant by RIA (Dechaud etal. 1989). It has been reported that the ammonium sulfate concentration is critical to proper precipitation of SHBG only, a parameter which should be accurately tested in each laboratory in order to avoid precipitation of albumin as well and underestimation of bioavailable testosterone (Davies etal. 2002).

When properly done, bioavailable testosterone correlates quite well with total testosterone and calculated free testosterone. The estimation ofbioavailable testosterone might be of value in clinical conditions of mild hypogonadism accompanied by increased SHBG and possibly reduced concentrations of serum albumin, such as in late-onset hypogonadism of the ageing male (Wheeler 1995;Morley etal. 2002).

21.4.4.6 Calculated free testosterone

The classical methods for free and bioavailable testosterone measurement reported above, i.e. equilibrium dialysis, ultrafiltration and ammonium sulfate precipitation are too cumbersome for clinical routine. This is the main reason why "analog" methods became so popular. A recent study compared the direct immunoassay of free testosterone by a labeled analog with calculation of free testosterone from total testosterone and immunoassayed SHBG concentrations, bioavailable testosterone assessed by ammonium sulphate precipitation and the free androgen index (ratio 100T/iSHBG). Using sera with a wide range of SHBG capacities, the study showed that calculated free testosterone (FT) values were almost identical to the values obtained by equilibrium dialysis except in sera from pregnant women, which contain high levels of saturated SHBG so that SHBG as determined by immunoassay overestimates its actual binding capacity (Vermeulen et al. 1999). The FT value is obtained from total testosterone and immunoassayable SHBG, assuming a constant albumin concentration and considering the equilibrium constants (K) of testosterone binding to SHBG and to albumin.

It was suggested that FT appears to be a rapid, simple, and reliable index of bioavailable testosterone, comparable to dialysis and suitable for clinical routine except in pregnancy. The formula and some examples of how to calculate FT starting from total testosterone and SHBG levels determined by immunoassay are available at http://www.issam.ch/freetesto.htm.

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