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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
What to Measure: Testosterone or Free Testosterone?
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<blockquote data-quote="madman" data-source="post: 223900" data-attributes="member: 13851"><p>No.</p><p></p><p>You should still be using what would be considered the most accurate assays for testing free testosterone such as the gold standard Equilibrium Dialysis or Ultrafiltration (next best), especially in cases of altered SHBG.</p><p></p><p>Use the same lab (reliable) and same assay (most accurate) every time you have blood work done.</p><p></p><p>For the time being until the limitations of testing free testosterone by (ED/UF) are addressed, you will have to put faith in the lab you are using.</p><p></p><p></p><p><em><strong>* Limitations of using free testosterone by equilibrium dialysis and calculated free testosterone concentrations in practice are the <u>lack of assay standardization, an accuracy-based quality control program, and a harmonized reference range</u>. Until these limitations are addressed, free testosterone by equilibrium dialysis and calculated free testosterone should use reference ranges established by individual laboratories or their specific assay method</strong></em></p><p></p><p><strong><em>*Assays that are standardized are designed to provide accurate results, <u>traceable to</u> <u>“true” value-assigned certified reference materials and gold-standard reference methods</u>. Results obtained using standardized methods can be compared across assays, institutions, populations, and past and future test results, thereby improving diagnosis, treatment, and outcomes of patients</em></strong></p><p><strong><em></em></strong></p><p><strong><em></em></strong></p><p><strong><em></em></strong></p><p><strong><em></em></strong></p><p><strong><em>post #4</em></strong></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/cdcs-clinical-standardization-programs-free-testosterone-soon-to-be.23242/[/URL]</p><p></p><p><strong>28:50-32:38</strong></p><p><strong></strong></p><p><strong>(29:20-30:05)</strong><em> <strong>We are working with different organizations on developing reference intervals that are based on certified assays. So we did already that successfully for testosterone.</strong></em></p><p><em><strong></strong></em></p><p><em><strong>We are currently working with some groups (research groups) on free testosterone reference intervals.</strong></em></p><p>[ATTACH=full]21803[/ATTACH]</p><p></p><p></p><p></p><p></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/measurement-of-ft-in-serum-using-ed-coupled-with-id-uhplc-ms-ms.23066/[/URL]</p><p></p><p><em><strong>*Measuring FT is technically challenging and shows high variability. <u>The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography-tandem mass spectrometry (ID-UHPLC-MS/MS</u>).</strong></em></p><p></p><p></p><p></p><p></p><p><strong><em>post #38</em></strong></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/cdc-standardized-total-t-and-estradiol-tests-and-soon-to-be-free-testosterone.17329/[/URL]</p><p></p><p>CDC Hormone Standardization Program (CDC HoSt) Certified Free Testosterone Procedures?</p><p></p><p>Sooner or later and SHBG to boot!</p><p></p><p>When the time comes these will be the only free testosterone assays I will recommend for men to use/rely upon when getting blood work done (pre/post-TRT).</p><p></p><p></p><p><strong>*a lab/assay that is certified by the CDC's HoSt Program</strong></p><p>[ATTACH=full]21804[/ATTACH]</p><p></p><p>The above applies to TT, estradiol, and soon enough free testosterone and SHBG!</p><p></p><p></p><p></p><p><strong><em>*Assays that are standardized are designed to provide accurate results,<u> traceable to “true” value-assigned certified reference materials and gold-standard reference methods</u>. Results obtained using standardized methods can be compared across assays, institutions, populations, and past and future test results, thereby improving diagnosis, treatment, and outcomes of patients</em></strong></p><p><strong><em></em></strong></p><p><strong><em>* Limitations of using free testosterone by equilibrium dialysis and calculated free testosterone concentrations in practice are the <u>lack of assay standardization, an accuracy-based quality control program, and a harmonized reference range</u>. Until these limitations are addressed, free testosterone by equilibrium dialysis and calculated free testosterone should use reference ranges established by individual laboratories or their specific assay method</em></strong></p><p><strong><em></em></strong></p><p><strong><em></em></strong></p><p><strong><em></em></strong></p><p><strong><em></em></strong></p><p><strong><em>post #8</em></strong></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/testosterone-deficiency-in-men.24722/[/URL]</p><p></p><p><em><strong>*Free testosterone concentration is ideally measured using the equilibrium dialysis method, performed under standardized conditions.1,31</strong></em></p><p><em><strong></strong></em></p><p><em><strong>*Direct tracer analog methods for measuring free testosterone concentrations are inaccurate, and therefore, their use is not recommended.35</strong></em></p><p></p><p><strong><em>*equations that are based on a linear model of testosterone’s binding to SHBG <u>assume a fixed binding affinity (approximately 1 nM)</u>31 and ignore the competing presence of other sex steroids, such as dihydrotestosterone and estradiol</em></strong></p><p></p><p><em><strong>*Recent studies using modern biophysical techniques have suggested that the binding of testosterone and estradiol to an SHBG dimer is a dynamic process that involves allosteric interactions between binding sites on each of the 2 SHBG monomers such that the<u> binding affinities of the 2 sites are not equivalent</u>.36,39</strong> <strong>The binding of a ligand to the first monomer influences the conformational and energetic states of both the monomers.39 The estimation of free testosterone concentration based on an ensemble allosteric model provides a close approximation of concentrations measured using equilibrium dialysis36;</strong></em></p><p><em><strong></strong></em></p><p><em><strong>*the computations of free testosterone concentrations using the ensemble allostery model can be obtained at <a href="https://tru-t.org/" target="_blank">TruT Free Testosterone Calculator by FPT</a></strong></em></p><p><em><strong></strong></em></p><p><em><strong>*Because of <u>dynamic changes in the binding affinity of SHBG upon ligand binding</u>, depending on the ligand and SHBG concentrations, <u>no equation can accurately estimate free testosterone concentration under all conditions</u>.39</strong></em></p><p></p><p></p><p></p><p></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/improving-diagnostic-accuracy-and-treatment-decisions-in-men-with-hypogonadism.24968/[/URL]</p><p></p><p><strong>24:05-37:00 (video)</strong></p><p><strong></strong></p><p><strong>28:36-31:58 (cFTV/cFTZ)</strong></p><p><strong></strong></p><p><strong>Shalender Bhasin :</strong></p><p><strong></strong></p><p><strong><em>*the calculated free testosterone is based on the simplistic notion that total testosterone is linearly related to free testosterone concentration and so you can compute total testosterone if you know the binding affinity and the concentrations of SHBG and albumin discounting orosomucoid and cortisol binding globulin and many people just ignore albumin because of the <u>perception that albumin-bound testosterone remains relatively in constant</u></em> </strong></p><p><strong></strong></p><p><strong><em>*and there are some empirically derived equations</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*and I'll show you that the linear equations such as the Vermeulens equation that is based on the <u>simplistic idea that one molecule of testosterone binds to one molecule of SHBG at a single binding site with a fixed binding affinity of 1 nanomolar (nM) to form a testosterone SHBG complex</u></em></strong></p><p><strong><em></em></strong></p><p><strong><em>*what are studies have shown is that testosterone binding to SHBG and we have shown similar data with estradiol which I'll show you in a minute</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*and that this binding is a <u>multi-step dynamic process</u></em></strong></p><p><strong><em></em></strong></p><p><strong><em>*so SHBG circulates as a dimer in circulation and this dimer is very tightly bound and it takes 8 nanomolar (nM) to dissociate the dimer but upon binding of the first testosterone to the first binding pocket in the first monomer there's a <u>change in conformation of the first monomer</u> as well as <u>change in the</u> <u>conformation of the second monomer</u> due to the<u> allosteric interaction between the two binding pockets</u> such that the binding of testosterone to the second binding pocket in the second monomer occurs with <u>a different binding affinity and this process is very dynamic so the Kd is not fixed</u> therefore the linear equations such as the Vermeulen equation and others that <u>assume a fixed Kd are just conceptually wrong and our data show it clearly that this model is completely wrong</u></em></strong></p><p><strong><em></em></strong></p><p><strong><em>*<u>so the Vermeulen model or most of the linear equations underestimate free testosterone concentrations</u> and the ensemble allosteric model (EAM) based upon our experimental data matches total testosterone within the range in which we have <u>validated</u> this</em></strong></p><p><strong><em></em></strong></p><p><strong><em>*so let me show you some additional data which persuade me that <u>no equation can ever be an accurate measure of the free testosterone at all levels of SHBG, testosterone, estradiol, DHT, and albumin under all conditions in men in women</u></em></strong></p><p></p><p></p><p></p><p></p><p>My reply from an earlier thread:</p><p></p><p><strong><em>post #16</em></strong></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/allosterically-coupled-multi-site-binding-of-t-to-human-serum-albumin.22102/[/URL]</p><p></p><p>The EAM appears to be an accurate and testable model for calculating free testosterone levels, but this model needs further validation in large populations.</p><p></p><p>This will be a part of the ongoing phase II.</p><p></p><p></p><p></p><p></p><p><strong><em>post #14</em></strong></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/improving-diagnostic-accuracy-and-treatment-decisions-in-men-with-hypogonadism.24968/#post-219173[/URL]</p><p></p><p></p><p>As I have stated numerous times on the forum!</p><p></p><p>Patiently waiting on the completion of Phase II for the TruT (cFTZ) Algorithm let alone standardization and harmonized reference ranges for Free testosterone which is in the works as we speak.</p><p></p><p>Phase II should be coming to an end soon enough.</p><p></p><p>8 years in 2014-2022.</p><p></p><p>Ravi let alone Shalender is confident about the validity/soon-to-be commercialization of the TruT Algorithm.</p><p></p><p>It's going to happen.</p><p></p><p>He is already pushing the use of the newer calculated method! <strong>(<a href="https://www.excelmale.com/forum/threads/testosterone-deficiency-in-men.24722/" target="_blank">Testosterone Deficiency in Men</a>)</strong></p><p></p><p>Much going on behind the scenes and more to come.</p><p></p><p></p><p></p><p><img src="https://www.excelmale.com/metadatalogo.png" class="bbImage" alt="" data-url="https://www.excelmale.com/metadatalogo.png" style="width:623px;" /></p><p></p><p></p><p></p><p></p><p>One of Bhasin's current papers.</p><p></p><p>[URL unfurl="true"]https://www.excelmale.com/forum/threads/improving-diagnostic-accuracy-and-treatment-decisions-in-men-with-hypogonadism.24968/[/URL]</p><p></p><p><em><strong>*<u>Free testosterone concentration is ideally measured using the equilibrium dialysis method, performed under standardized conditions</u>.1,31<u> Direct tracer analog methods for measuring free testosterone concentrations are inaccurate, and therefore, their use is not recommended</u>.35 Although several equations to estimate free testosterone concentration from total testosterone, SHBG, and albumin concentrations have been published,36-38 the estimation of free testosterone concentration performed using these equations are predicated upon accurate measurements of total testosterone, SHBG, and albumin concentrations.31,35 <u>Furthermore, equations that are based on a linear model of testosterone’s binding to SHBG assume a fixed binding affinity (approximately 1 nM)31 and ignore the competing presence of other sex steroids, such as dihydrotestosterone and estradiol</u>.</strong></em></p><p><em><strong></strong></em></p><p><em><strong>*Recent studies using modern biophysical techniques have suggested that the <u>binding of testosterone and estradiol to an SHBG dimer is a dynamic process that involves allosteric interactions between binding sites on each of the 2 SHBG monomers such that the binding affinities of the 2 sites are not equivalent</u>.36,39 <u>The binding of a ligand to the first monomer influences the conformational and energetic states of both the monomers</u>.39 The estimation of free testosterone concentration based on an ensemble allosteric model provides a close approximation of concentrations measured using equilibrium dialysis36;</strong></em></p><p><em><strong></strong></em></p><p><em><strong>*<u>the computations of free testosterone concentrations using the ensemble allostery model can be obtained at <a href="https://tru-t.org/" target="_blank">TruT Free Testosterone Calculator by FPT</a></u>. Because of dynamic changes in the binding affinity of SHBG upon ligand binding, depending on the ligand and SHBG concentrations, <u>no equation can accurately estimate free testosterone concentration under all conditions</u>.39</strong></em></p><p></p><p></p><p></p><p><strong>Phase II: Research and Commercialization of TruT Algorithm (Sep.15, 2014-May 31, 2022)</strong></p><p><strong>[ATTACH=full]21805[/ATTACH]</strong></p><p></p><p></p><p></p><p></p><p>They have most likely wrapped up Phase II and it is just a matter of time before the data comes out.</p><p></p><p>For the time being, do what you feel is best for you.</p><p></p><p>Just make sure to steer clear of using/relying upon the piss poor direct immunoassay!</p></blockquote><p></p>
[QUOTE="madman, post: 223900, member: 13851"] No. You should still be using what would be considered the most accurate assays for testing free testosterone such as the gold standard Equilibrium Dialysis or Ultrafiltration (next best), especially in cases of altered SHBG. Use the same lab (reliable) and same assay (most accurate) every time you have blood work done. For the time being until the limitations of testing free testosterone by (ED/UF) are addressed, you will have to put faith in the lab you are using. [I][B]* Limitations of using free testosterone by equilibrium dialysis and calculated free testosterone concentrations in practice are the [U]lack of assay standardization, an accuracy-based quality control program, and a harmonized reference range[/U]. Until these limitations are addressed, free testosterone by equilibrium dialysis and calculated free testosterone should use reference ranges established by individual laboratories or their specific assay method[/B][/I] [B][I]*Assays that are standardized are designed to provide accurate results, [U]traceable to[/U] [U]“true” value-assigned certified reference materials and gold-standard reference methods[/U]. Results obtained using standardized methods can be compared across assays, institutions, populations, and past and future test results, thereby improving diagnosis, treatment, and outcomes of patients post #4[/I][/B] [URL unfurl="true"]https://www.excelmale.com/forum/threads/cdcs-clinical-standardization-programs-free-testosterone-soon-to-be.23242/[/URL] [B]28:50-32:38 (29:20-30:05)[/B][I] [B]We are working with different organizations on developing reference intervals that are based on certified assays. So we did already that successfully for testosterone. We are currently working with some groups (research groups) on free testosterone reference intervals.[/B][/I] [ATTACH type="full" alt="Screenshot (13088).png"]21803[/ATTACH] [URL unfurl="true"]https://www.excelmale.com/forum/threads/measurement-of-ft-in-serum-using-ed-coupled-with-id-uhplc-ms-ms.23066/[/URL] [I][B]*Measuring FT is technically challenging and shows high variability. [U]The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography-tandem mass spectrometry (ID-UHPLC-MS/MS[/U]).[/B][/I] [B][I]post #38[/I][/B] [URL unfurl="true"]https://www.excelmale.com/forum/threads/cdc-standardized-total-t-and-estradiol-tests-and-soon-to-be-free-testosterone.17329/[/URL] CDC Hormone Standardization Program (CDC HoSt) Certified Free Testosterone Procedures? Sooner or later and SHBG to boot! When the time comes these will be the only free testosterone assays I will recommend for men to use/rely upon when getting blood work done (pre/post-TRT). [B]*a lab/assay that is certified by the CDC's HoSt Program[/B] [ATTACH type="full" alt="Screenshot (13089).png"]21804[/ATTACH] The above applies to TT, estradiol, and soon enough free testosterone and SHBG! [B][I]*Assays that are standardized are designed to provide accurate results,[U] traceable to “true” value-assigned certified reference materials and gold-standard reference methods[/U]. Results obtained using standardized methods can be compared across assays, institutions, populations, and past and future test results, thereby improving diagnosis, treatment, and outcomes of patients * Limitations of using free testosterone by equilibrium dialysis and calculated free testosterone concentrations in practice are the [U]lack of assay standardization, an accuracy-based quality control program, and a harmonized reference range[/U]. Until these limitations are addressed, free testosterone by equilibrium dialysis and calculated free testosterone should use reference ranges established by individual laboratories or their specific assay method post #8[/I][/B] [URL unfurl="true"]https://www.excelmale.com/forum/threads/testosterone-deficiency-in-men.24722/[/URL] [I][B]*Free testosterone concentration is ideally measured using the equilibrium dialysis method, performed under standardized conditions.1,31 *Direct tracer analog methods for measuring free testosterone concentrations are inaccurate, and therefore, their use is not recommended.35[/B][/I] [B][I]*equations that are based on a linear model of testosterone’s binding to SHBG [U]assume a fixed binding affinity (approximately 1 nM)[/U]31 and ignore the competing presence of other sex steroids, such as dihydrotestosterone and estradiol[/I][/B] [I][B]*Recent studies using modern biophysical techniques have suggested that the binding of testosterone and estradiol to an SHBG dimer is a dynamic process that involves allosteric interactions between binding sites on each of the 2 SHBG monomers such that the[U] binding affinities of the 2 sites are not equivalent[/U].36,39[/B] [B]The binding of a ligand to the first monomer influences the conformational and energetic states of both the monomers.39 The estimation of free testosterone concentration based on an ensemble allosteric model provides a close approximation of concentrations measured using equilibrium dialysis36; *the computations of free testosterone concentrations using the ensemble allostery model can be obtained at [URL='https://tru-t.org/']TruT Free Testosterone Calculator by FPT[/URL] *Because of [U]dynamic changes in the binding affinity of SHBG upon ligand binding[/U], depending on the ligand and SHBG concentrations, [U]no equation can accurately estimate free testosterone concentration under all conditions[/U].39[/B][/I] [URL unfurl="true"]https://www.excelmale.com/forum/threads/improving-diagnostic-accuracy-and-treatment-decisions-in-men-with-hypogonadism.24968/[/URL] [B]24:05-37:00 (video) 28:36-31:58 (cFTV/cFTZ) Shalender Bhasin : [I]*the calculated free testosterone is based on the simplistic notion that total testosterone is linearly related to free testosterone concentration and so you can compute total testosterone if you know the binding affinity and the concentrations of SHBG and albumin discounting orosomucoid and cortisol binding globulin and many people just ignore albumin because of the [U]perception that albumin-bound testosterone remains relatively in constant[/U][/I] [I]*and there are some empirically derived equations *and I'll show you that the linear equations such as the Vermeulens equation that is based on the [U]simplistic idea that one molecule of testosterone binds to one molecule of SHBG at a single binding site with a fixed binding affinity of 1 nanomolar (nM) to form a testosterone SHBG complex[/U] *what are studies have shown is that testosterone binding to SHBG and we have shown similar data with estradiol which I'll show you in a minute *and that this binding is a [U]multi-step dynamic process[/U] *so SHBG circulates as a dimer in circulation and this dimer is very tightly bound and it takes 8 nanomolar (nM) to dissociate the dimer but upon binding of the first testosterone to the first binding pocket in the first monomer there's a [U]change in conformation of the first monomer[/U] as well as [U]change in the[/U] [U]conformation of the second monomer[/U] due to the[U] allosteric interaction between the two binding pockets[/U] such that the binding of testosterone to the second binding pocket in the second monomer occurs with [U]a different binding affinity and this process is very dynamic so the Kd is not fixed[/U] therefore the linear equations such as the Vermeulen equation and others that [U]assume a fixed Kd are just conceptually wrong and our data show it clearly that this model is completely wrong[/U] *[U]so the Vermeulen model or most of the linear equations underestimate free testosterone concentrations[/U] and the ensemble allosteric model (EAM) based upon our experimental data matches total testosterone within the range in which we have [U]validated[/U] this *so let me show you some additional data which persuade me that [U]no equation can ever be an accurate measure of the free testosterone at all levels of SHBG, testosterone, estradiol, DHT, and albumin under all conditions in men in women[/U][/I][/B] My reply from an earlier thread: [B][I]post #16[/I][/B] [URL unfurl="true"]https://www.excelmale.com/forum/threads/allosterically-coupled-multi-site-binding-of-t-to-human-serum-albumin.22102/[/URL] The EAM appears to be an accurate and testable model for calculating free testosterone levels, but this model needs further validation in large populations. This will be a part of the ongoing phase II. [B][I]post #14[/I][/B] [URL unfurl="true"]https://www.excelmale.com/forum/threads/improving-diagnostic-accuracy-and-treatment-decisions-in-men-with-hypogonadism.24968/#post-219173[/URL] As I have stated numerous times on the forum! Patiently waiting on the completion of Phase II for the TruT (cFTZ) Algorithm let alone standardization and harmonized reference ranges for Free testosterone which is in the works as we speak. Phase II should be coming to an end soon enough. 8 years in 2014-2022. Ravi let alone Shalender is confident about the validity/soon-to-be commercialization of the TruT Algorithm. It's going to happen. He is already pushing the use of the newer calculated method! [B]([URL="https://www.excelmale.com/forum/threads/testosterone-deficiency-in-men.24722/"]Testosterone Deficiency in Men[/URL])[/B] Much going on behind the scenes and more to come. [IMG='width:623px; width="623px"']https://www.excelmale.com/metadatalogo.png[/IMG] One of Bhasin's current papers. [URL unfurl="true"]https://www.excelmale.com/forum/threads/improving-diagnostic-accuracy-and-treatment-decisions-in-men-with-hypogonadism.24968/[/URL] [I][B]*[U]Free testosterone concentration is ideally measured using the equilibrium dialysis method, performed under standardized conditions[/U].1,31[U] Direct tracer analog methods for measuring free testosterone concentrations are inaccurate, and therefore, their use is not recommended[/U].35 Although several equations to estimate free testosterone concentration from total testosterone, SHBG, and albumin concentrations have been published,36-38 the estimation of free testosterone concentration performed using these equations are predicated upon accurate measurements of total testosterone, SHBG, and albumin concentrations.31,35 [U]Furthermore, equations that are based on a linear model of testosterone’s binding to SHBG assume a fixed binding affinity (approximately 1 nM)31 and ignore the competing presence of other sex steroids, such as dihydrotestosterone and estradiol[/U]. *Recent studies using modern biophysical techniques have suggested that the [U]binding of testosterone and estradiol to an SHBG dimer is a dynamic process that involves allosteric interactions between binding sites on each of the 2 SHBG monomers such that the binding affinities of the 2 sites are not equivalent[/U].36,39 [U]The binding of a ligand to the first monomer influences the conformational and energetic states of both the monomers[/U].39 The estimation of free testosterone concentration based on an ensemble allosteric model provides a close approximation of concentrations measured using equilibrium dialysis36; *[U]the computations of free testosterone concentrations using the ensemble allostery model can be obtained at [URL='https://tru-t.org/']TruT Free Testosterone Calculator by FPT[/URL][/U]. Because of dynamic changes in the binding affinity of SHBG upon ligand binding, depending on the ligand and SHBG concentrations, [U]no equation can accurately estimate free testosterone concentration under all conditions[/U].39[/B][/I] [B]Phase II: Research and Commercialization of TruT Algorithm (Sep.15, 2014-May 31, 2022) [ATTACH type="full" alt="Screenshot (13090).png"]21805[/ATTACH][/B] They have most likely wrapped up Phase II and it is just a matter of time before the data comes out. For the time being, do what you feel is best for you. Just make sure to steer clear of using/relying upon the piss poor direct immunoassay! [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
What to Measure: Testosterone or Free Testosterone?
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