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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
Reassessing Free-Testosterone Calculation by Liquid Chromatography–Tandem Mass Spectrometry Direct Equilibrium Dialysis
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<blockquote data-quote="tareload" data-source="post: 189340"><p>[URL unfurl="true"]https://academic.oup.com/edrv/article/38/4/297/4071740[/URL]</p><p></p><p>Widely used model-based formulae by Södergård et al. (21), Vermeulen et al. (22), and Mazer (23) incorporate two major sources of potential systematic error: assumptions about the stoichiometry and the binding affinity of testosterone for SHBG. Although the review attributes the inaccuracies of model-based formulae to their “linearity,” the discrepancies arise for a different reason: the mistaken assumptions on the stoichiometry of testosterone binding to SHBG. Prior to the crystal structure of SHBG in 2001 (24), the SHBG homodimer was believed to bind only a single testosterone molecule at dimerization junction (25), an assumption incorporated into all model-based FT formulae. However, the SHBG crystal structure showed that each monomer had a testosterone binding pocket so that the homodimer binds two, not one, testosterone molecules. Correcting this erroneous stoichiometry incorporated into the model-based binding equations reduces the marked deviations of model-based formulae from laboratory measured FT (26, 27). Another untested assumption of model-based formulae is the substitution of gravimetric measurement of SHBG by immunoassay—which differ between immunoassay methods—for SHBG binding capacity, which the binding equations actually invoke. Finally, the impact of genetic polymorphisms or acquired disease-related changes in SHBG binding affinity cannot be incorporated into the model-based FT formulae.</p><p></p><p>The review highlights a biophysical model by Zakharov et al. (28) for testosterone binding to SHBG with a novel but incompletely disclosed formula, patented by review coauthors and not yet subject to independent evaluation. The Zakharov biophysical model is a refinement of the equilibrium binding models by fitting two allosterically linked binding sites with distinct affinities, instead of two binding sites with the same affinity per SHBG monomer.<strong> Hence, although this work refines the understanding of the molecular binding of testosterone to SHBG, this minor variant of a model-based formula does not overcome the limitation of an obligatory requirement for plug-in binding affinities. I</strong>mprovement by this thermodynamic model is at least partly attributable to its incorporation of the correct testosterone binding stoichiometry to SHBG<strong>. More troublesome problems arise for model-based FT formulae in their requirement for plug-in binding affinities of testosterone for SHBG, which are highly influential and responsible for much of the deviations of model-based formulae from laboratory-measured FT. Measuring such binding affinities, assumed to be population-wide and invariant, is as exacting as dialysis-based direct FT measurement; yet there is a fivefold variation among such estimates used in various equations (23).</strong></p><p></p><p>The alternative, assumption-free fully empirical equations are given scant coverage denying them a fair hearing. These circumvent pitfalls arising from the assumptions requiring use of plug-in estimates for testosterone’s stoichiometry and binding affinity to SHBG. The fully empirical equations are created by regression of dialysis-based laboratory measured FT on measured serum testosterone and SHBG in the same samples using large databases, a methodology open to updating with new data. The most recent empirical formulae derived from a large data set (>4000 serum samples) has been subsequently verified in a different large data set (>2000 serum samples) from another laboratory (27, 29)<strong>. In both studies, direct head-to-head testing showed that the model-based formulae consistently overestimate FT (27, 29, 30), a finding confirmed independently by others (26, 31–34). Curiously, Zakharov FT calculations produce even higher results than the Vermeulen equation making further independent evaluations of its validity essential. In that context, it is premature for the review to cite a lower confidence limit for calculated FT when the studies on which it is based have given rise to widely differing reference ranges for serum testosterone having reported a lower 2.5th centile of 348.3 ng/dL in one report (35) but subsequently as a 40% lower one (209 ng/dL) (36).</strong></p></blockquote><p></p>
[QUOTE="tareload, post: 189340"] [URL unfurl="true"]https://academic.oup.com/edrv/article/38/4/297/4071740[/URL] Widely used model-based formulae by Södergård et al. (21), Vermeulen et al. (22), and Mazer (23) incorporate two major sources of potential systematic error: assumptions about the stoichiometry and the binding affinity of testosterone for SHBG. Although the review attributes the inaccuracies of model-based formulae to their “linearity,” the discrepancies arise for a different reason: the mistaken assumptions on the stoichiometry of testosterone binding to SHBG. Prior to the crystal structure of SHBG in 2001 (24), the SHBG homodimer was believed to bind only a single testosterone molecule at dimerization junction (25), an assumption incorporated into all model-based FT formulae. However, the SHBG crystal structure showed that each monomer had a testosterone binding pocket so that the homodimer binds two, not one, testosterone molecules. Correcting this erroneous stoichiometry incorporated into the model-based binding equations reduces the marked deviations of model-based formulae from laboratory measured FT (26, 27). Another untested assumption of model-based formulae is the substitution of gravimetric measurement of SHBG by immunoassay—which differ between immunoassay methods—for SHBG binding capacity, which the binding equations actually invoke. Finally, the impact of genetic polymorphisms or acquired disease-related changes in SHBG binding affinity cannot be incorporated into the model-based FT formulae. The review highlights a biophysical model by Zakharov et al. (28) for testosterone binding to SHBG with a novel but incompletely disclosed formula, patented by review coauthors and not yet subject to independent evaluation. The Zakharov biophysical model is a refinement of the equilibrium binding models by fitting two allosterically linked binding sites with distinct affinities, instead of two binding sites with the same affinity per SHBG monomer.[B] Hence, although this work refines the understanding of the molecular binding of testosterone to SHBG, this minor variant of a model-based formula does not overcome the limitation of an obligatory requirement for plug-in binding affinities. I[/B]mprovement by this thermodynamic model is at least partly attributable to its incorporation of the correct testosterone binding stoichiometry to SHBG[B]. More troublesome problems arise for model-based FT formulae in their requirement for plug-in binding affinities of testosterone for SHBG, which are highly influential and responsible for much of the deviations of model-based formulae from laboratory-measured FT. Measuring such binding affinities, assumed to be population-wide and invariant, is as exacting as dialysis-based direct FT measurement; yet there is a fivefold variation among such estimates used in various equations (23).[/B] The alternative, assumption-free fully empirical equations are given scant coverage denying them a fair hearing. These circumvent pitfalls arising from the assumptions requiring use of plug-in estimates for testosterone’s stoichiometry and binding affinity to SHBG. The fully empirical equations are created by regression of dialysis-based laboratory measured FT on measured serum testosterone and SHBG in the same samples using large databases, a methodology open to updating with new data. The most recent empirical formulae derived from a large data set (>4000 serum samples) has been subsequently verified in a different large data set (>2000 serum samples) from another laboratory (27, 29)[B]. In both studies, direct head-to-head testing showed that the model-based formulae consistently overestimate FT (27, 29, 30), a finding confirmed independently by others (26, 31–34). Curiously, Zakharov FT calculations produce even higher results than the Vermeulen equation making further independent evaluations of its validity essential. In that context, it is premature for the review to cite a lower confidence limit for calculated FT when the studies on which it is based have given rise to widely differing reference ranges for serum testosterone having reported a lower 2.5th centile of 348.3 ng/dL in one report (35) but subsequently as a 40% lower one (209 ng/dL) (36).[/B] [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
Reassessing Free-Testosterone Calculation by Liquid Chromatography–Tandem Mass Spectrometry Direct Equilibrium Dialysis
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