Introduction This free testosterone (FTe) assay is routinely applied to whole serum. In whole male serum 1.5 to 2.0% of total testosterone (TTe) is FTe. (The remainder is bound to Te binding serum proteins.) The FTe assay is based on a radiolabeled Te analogue (a Te conjugate) that competes with unlabeled Te for binding to a Te-specific antibody. Two questions were asked about the assay. First, how does the assay respond when only protein-bound Te and TTe are diluted while FTe is constant? Second, how does the assay respond when FTe replaces protein-bound Te while TTe remains constant? Analogue-based FTe estimates and TTe determinations were compared.
Materials and Methods Equilibrium dialysis (Nichols Institute Diagnostics) was applied to normal adult male serum. The free Te assay was applied to dialysate. A total Te assay was applied to retentate. Te was added to an aliquot of dialysate until total Te matched retentate total Te. One aliquot of serum retentate was progressively diluted with serum dialysate (no Te added). Thus, protein-bound and total Te were decreased while free Te remained constant. Another aliquot of serum retentate was progressively diluted with the dialysate containing added Te. Thus, protein-bound Te decreased and FTe increased while TTe remained constant. The FTe RIA and the TTe RIA (both from Diagnostic Products Company, Los Angeles, CA) were applied to all Te solutions.
Results Free Te was not detected in serum dialysate. When TTe and protein-bound Te were progressively reduced while free Te was constant, TTe determinations progressively declined 95%, from 581 to 29 ng/dL. The median was 167 ng/dL. The analogue-based FTe determinations also progressively declined ≈95% from 1.28 to < 0.06 ng/dL. The median was 0.16 ng/dL. FTe and TTe determinations were closely correlated (r2 .98). When protein-bound Te was progressively replaced with FTe while total Te was constant (648 ng/dL ± 14), free Te determinations were similarly constant (1.48 ng/dL ± 0.02).
Conclusions These analogue-based free Te estimates were not traceable to serum free Te concentrations. Free Te determinations were traceable to total Te concentrations and total Te concentrations only. The forms of Te tracked and the concentrations of Te detected by this assay provide direct evidence of protein-bound Te cross-reactivity. No evidence was found to support the concept that this free Te RIA tracks or detects serum free Te.
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