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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
Allosterically coupled multi-site binding of T to human serum albumin
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<blockquote data-quote="madman" data-source="post: 190020" data-attributes="member: 13851"><p>Much more going on here than SHBG:T binding when it comes to cFT!</p><p></p><p>Patiently waiting on the completion of Phase II for the TruT (cFTZ) Algorithm</p><p></p><p></p><p></p><p></p><p><strong>R44G045011 <span style="color: rgb(184, 49, 47)">(JASUJA, RAVI)</span></strong></p><p><strong>NIH/NIA</strong></p><p><strong>Phase II: <span style="color: rgb(184, 49, 47)">Research and Commercialization of TruT Algorithm</span></strong></p><p><strong>Role: Principal Investigator</strong></p><p><strong></strong></p><p><strong>Sep 15, 2017 - <span style="color: rgb(184, 49, 47)">May 31, 2021</span></strong></p><p></p><p>[ATTACH=full]11404[/ATTACH]</p><p></p><p></p><p></p><p></p><p></p><p></p><p><strong>A Reappraisal of Testosterone's Binding in Circulation: <span style="color: rgb(184, 49, 47)">Physiological and Clinical Implications </span>(2017)</strong></p><p><span style="color: rgb(44, 130, 201)">Anna L Goldman,</span><span style="color: rgb(184, 49, 47)"> <strong>Shalender Bhasin</strong></span><span style="color: rgb(44, 130, 201)">, Frederick C W Wu, Meenakshi Krishna, Alvin M Matsumoto, </span><span style="color: rgb(184, 49, 47)"><strong>Ravi Jasuja</strong></span></p><p></p><p></p><p></p><p></p><p><strong>Abstract</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)"><u>In the circulation, testosterone and other sex hormones are bound to binding proteins, which play an important role in regulating their transport, distribution, metabolism, and biological activity</u>.</span></em> According to the free hormone hypothesis, which has been debated extensively, only the unbound or free fraction is biologically active in target tissues. <em><span style="color: rgb(184, 49, 47)">Consequently,<u> accurate determination of the partitioning of testosterone between bound and free fractions is central to our understanding of how it's delivery to the target tissues and biological activity are regulated</u> and consequently to the diagnosis and treatment of androgen disorders in men and women.</span></em> <strong><em><span style="color: rgb(184, 49, 47)">Here, we present a historical perspective on the evolution of our understanding of the binding of testosterone to circulating binding proteins. <u>On the basis of an appraisal of the literature as well as experimental data, we show that the assumptions of stoichiometry, binding dynamics, and the affinity of the prevailing models of testosterone binding to sex hormone-binding globulin and </u></span><span style="color: rgb(44, 130, 201)"><u>human serum albumin</u></span><span style="color: rgb(184, 49, 47)"><u> are not supported by published experimental data and are most likely inaccurate</u>.</span> </em></strong>This review offers some guiding principles for the application of free testosterone measurements in the diagnosis and treatment of patients with androgen disorders. The growing number of testosterone prescriptions and widely recognized problems with the direct measurement as well as the computation of free testosterone concentrations render this critical review timely and clinically relevant.</p><p></p><p></p><p></p><p></p><p><strong>Essential Points</strong></p><p></p><p><span style="color: rgb(184, 49, 47)">Most circulating testosterone is bound to its cognate binding proteins—sex hormone−binding globulin (SHBG), </span><span style="color: rgb(44, 130, 201)"><u>human serum albumin (HSA)</u></span><span style="color: rgb(184, 49, 47)">, cortisol-binding globulin, and orosomucoid; <u>these binding proteins play an important role in regulating the transport, tissue delivery, bioactivity, and metabolism of testosterone</u> </span></p><p><span style="color: rgb(184, 49, 47)"></span></p><p><span style="color: rgb(184, 49, 47)">The physiochemical characteristics and dynamics of the binding of testosterone to its binding proteins are poorly understood; <u>oversimplified assumptions of stoichiometry, binding dynamics, and binding affinity have contributed to the development of inaccurate linear binding models of testosterone to SHBG and </u></span><span style="color: rgb(44, 130, 201)"><u>HSA</u> </span></p><p></p><p><span style="color: rgb(184, 49, 47)"><u>The ensemble allosteric model of the binding of testosterone to SHBG developed from recent studies using modern biophysical techniques suggests that testosterone binding to SHBG is a complex, multistep process that involves interbinding site allostery </u></span></p><p></p><p><span style="color: rgb(44, 130, 201)"><u>The dynamics of the binding of testosterone to HSA</u></span><span style="color: rgb(184, 49, 47)"><u>, orosomucoid, and corticosteroid-binding globulin also require careful reexamination because the roles of these binding proteins in regulating circulating testosterone concentrations remain incompletely understood</u> </span></p><p><span style="color: rgb(184, 49, 47)"></span></p><p><span style="color: rgb(184, 49, 47)">I<u>f the free hormone hypothesis is correct (<em>i.e.,</em> only free testosterone is biologically active), accurate determination and harmonized reference ranges for free testosterone are necessary to diagnose androgen disorders in men and women</u></span></p><p><span style="color: rgb(184, 49, 47)"><u></u></span></p><p><span style="color: rgb(184, 49, 47)"><u>Methods for the measurement of free testosterone levels are fraught with potential problems, including poor precision, inaccuracy, and low specificity, and reliable assays are not readily available to practicing clinicians; therefore, </u></span><span style="color: rgb(44, 130, 201)"><u>algorithms based on valid binding models that can be used to estimate circulating free testosterone levels</u></span><span style="color: rgb(184, 49, 47)"><u> are needed to facilitate sound clinical decision making</u> </span></p><p><span style="color: rgb(184, 49, 47)"></span></p><p><span style="color: rgb(184, 49, 47)"></span></p><p><span style="color: rgb(184, 49, 47)"></span></p><p><span style="color: rgb(184, 49, 47)"></span></p><p><span style="color: rgb(184, 49, 47)"><em><u>Binding proteins in the peripheral circulation are important in regulating the transport, bioavailability, and metabolism of their cognate ligands, such as steroid hormones, fatty acids, vitamins, and drugs</u>. The major sex steroid hormones—testosterone, 5α-dihydrotestosterone, and 17β-estradiol—bind predominantly to sex hormone−binding globulin (SHBG) and to </em></span><span style="color: rgb(44, 130, 201)"><em>human serum albumin (HSA)</em></span><span style="color: rgb(184, 49, 47)"><em> and to a lesser extent to corticosteroid-binding globulin (CBG) and orosomucoid. SHBG, which is secreted by the liver, binds to testosterone with high affinity and is an important determinant of the distribution of circulating testosterone into its bound and free fractions (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">1</a>). </em></span><span style="color: rgb(44, 130, 201)"><em><u>HSA is one of the most abundant and versatile proteins in circulation; although it binds testosterone with lower affinity than SHBG does, its high binding capacity and high concentration allow it to buffer fluctuations in testosterone levels</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">1</a>).</em></span> The characteristics of testosterone binding to CBG and orosomucoid and the biological roles of these binding proteins in regulating testosterone bioavailability remain incompletely understood.</p><p></p><p></p><p></p><p></p><p><em><strong>Total testosterone</strong></em> <em><span style="color: rgb(184, 49, 47)">refers to the sum of the concentrations of protein-bound and unbound testosterone in circulation. The fraction of circulating testosterone that is unbound to any plasma protein is referred to as the</span></em> <em><span style="color: rgb(0, 0, 0)"><strong>free testosterone fraction</strong></span></em><strong>. </strong><span style="color: rgb(184, 49, 47)"><em>The term</em></span> <em><strong>bioavailable testosterone</strong></em> <span style="color: rgb(184, 49, 47)"><em>refers to the fraction of circulating testosterone that is not bound to SHBG and largely represents the sum of free testosterone plus </em></span><span style="color: rgb(44, 130, 201)"><em>HSA-bound testosterone</em></span> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 1</a>) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">2</a>); <span style="color: rgb(44, 130, 201)"><em><u>the term reflects the view that HSA-bound testosterone, which is bound with low affinity, can dissociate from HSA in the tissue capillaries and effectively be available for biological activity</u>.</em></span> <em><span style="color: rgb(184, 49, 47)">The free testosterone fraction can be measured directly by the equilibrium dialysis or ultrafiltration method or calculated from total testosterone, SHBG, and </span><span style="color: rgb(44, 130, 201)">HSA </span><span style="color: rgb(184, 49, 47)">concentrations using published mass action binding algorithms</span></em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3–6</a>). The bioavailable fraction can be measured using the ammonium sulfate precipitation method or the concanavalin A method, or it can be calculated from total testosterone, SHBG, and HSA concentrations (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">7</a>). Although the pioneers who originated the concept of bioavailable testosterone envisioned it as the sum of HSA-bound and unbound fractions of circulating testosterone (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">2</a>), the methods used to measure bioavailable testosterone concentrations, namely, the ammonium sulfate precipitation and concanavalin A methods, quantitate it as the non−SHBG-bound fraction of circulating testosterone, which approximates but is not equivalent to its original conceptualization as the sum of HSA-bound plus unbound testosterone levels (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">8</a>).</p><p></p><p>[ATTACH=full]11395[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">Partitioning of testosterone in the systemic circulation.</span> Circulating testosterone is bound tightly to SHBG<span style="color: rgb(26, 188, 156)"> (green = high-affinity binding) </span>and <span style="color: rgb(0, 0, 0)">weakly to</span><span style="color: rgb(44, 130, 201)"> albumin, </span><span style="color: rgb(0, 0, 0)">orosomucoid (ORM), and CBG </span><span style="color: rgb(44, 130, 201)">(blue = low-affinity binding)</span> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">11</a>). <span style="color: rgb(184, 49, 47)"><u>Only 1% to 4% of circulating testosterone is unbound or free</u>.</span> The combination of free and <span style="color: rgb(44, 130, 201)">albumin-bound testosterone</span> is also referred to as the “bioavailable testosterone” fraction. </strong></p><p></p><p></p><p></p><p></p><p><span style="color: rgb(184, 49, 47)"><em><u>The validity of calculated bioavailable and free testosterone is predicated on the accuracy of binding protein and testosterone concentrations and on the veracity of the assumptions of the association stoichiometry, binding affinities, and binding dynamics underlying the molecular binding model</u>.</em></span> The foundational assumptions about the relationship between testosterone and its binding proteins and estimates of the biophysical parameters of testosterone binding to its cognate binding proteins, upon which many extant algorithms for computing free testosterone are based, have undergone recent reappraisal and are discussed later. <span style="color: rgb(184, 49, 47)"><em><u>Data from the experimental studies performed in the 1960s and 1970s have been extrapolated without acknowledgment of the lack of experimental support for the underlying assumptions about linearity (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">9–11</a>) or of the methodological limitations described by the original authors</u>. <u>Collectively, these have led to an oversimplification of binding models based on somewhat erroneous assumptions of stoichiometry, binding affinity, and binding dynamics</u>. </em></span></p><p></p><p>The rapid growth of testosterone prescriptions during the last decade (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">12</a>) has refocused attention on the critical need for accurate determination of free testosterone in the diagnostic evaluation of men with a suspected androgen deficiency and for rational dosing and monitoring of testosterone replacement therapy. <em><span style="color: rgb(184, 49, 47)">Accordingly, an expository review of the published data and prevailing models of testosterone binding is timely. <u>Here, we present a historical perspective of the evolution of our understanding of the binding and bioavailability of testosterone. This review attempts to provide a comprehensive and critical appraisal of the prevailing models of testosterone binding to SHBG and</u></span><span style="color: rgb(44, 130, 201)"><u> HSA</u></span><span style="color: rgb(184, 49, 47)"><u>, the associated biophysical parameters, and their underlying assumptions and limitations</u>.</span></em> <em><span style="color: rgb(184, 49, 47)"><u>We discuss how recent advances in the computational and biophysical techniques have begun to unravel the multistep dynamics of testosterone binding to its cognate binding proteins, including the allosteric interactions between the testosterone binding sites on the SHBG dimer</u>. </span></em>This review also provides a contemporary perspective on the validity of the free hormone hypothesis and the clinical implications of these findings in the diagnosis, treatment, and monitoring of men with hypogonadism.</p><p></p><p></p><p></p><p></p><p><strong>Biology of Binding Proteins and Their Role in the Transport, Distribution, Metabolism, and Bioavailability of Testosterone</strong></p><p></p><p><span style="color: rgb(184, 49, 47)"><em>At least four structurally distinct binding proteins are known to bind testosterone in human circulation: SHBG, </em></span><span style="color: rgb(44, 130, 201)"><em>HSA</em></span><span style="color: rgb(184, 49, 47)"><em>, CBG, and orosomucoid. </em></span>Among these, SHBG has received the most attention because of its high binding affinity for testosterone. <em><span style="color: rgb(184, 49, 47)">T<u>hese binding proteins influence the tissue bioavailability and metabolic clearance rate of testosterone by regulating the amount of free testosterone available for biological action in the tissue</u>. The roles of</span><span style="color: rgb(44, 130, 201)"> HSA</span><span style="color: rgb(184, 49, 47)">, CBG, and orosomucoid in regulating testosterone’s bioavailability are less well understood, <u>and we do not know how disease states or conditions that may differentially alter the circulating concentrations of </u></span><u><span style="color: rgb(44, 130, 201)">HSA</span></u><span style="color: rgb(184, 49, 47)"><u>, CBG, and orosomucoid impact the binding of testosterone to SHBG</u>.</span></em> <em><span style="color: rgb(184, 49, 47)"><strong><u>Current computations of free and bioavailable testosterone account only for the potential impact of alterations in </u></strong></span><u><span style="color: rgb(44, 130, 201)"><strong>HSA</strong></span></u><span style="color: rgb(184, 49, 47)"><strong><u> and SHBG, ignoring CBG and orosomucoid and other potentially interacting proteins and steroid hormones</u></strong>.</span></em></p><p></p><p></p><p></p><p><strong>HSA</strong></p><p></p><p><em><span style="color: rgb(44, 130, 201)"><u>HSA is the most abundant protein in the human circulation, accounting for 60% of the total serum protein content and having a concentration of 30 to 50 g/L (450 to 750 µM) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">35</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">36</a>). From 33% to 54% of testosterone binds with low affinity to HSA, with an association constant of 2.0 to 4.1 × 104 L/mol at 37°C</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">5</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">37–39</a>). </span></em>Albumin Catania (580 Lys-Leu-Pro-COOH) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">40</a>) and albumin Roma (321 Glu-Lys) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">41</a>) are known variants that impact the affinity of HSA for testosterone; albumin Roma has a decreased affinity for testosterone, and it is unknown if albumin Catania has an increased or decreased affinity.</p><p></p><p>The high capacity of HSA for binding steroids is particularly highlighted during pregnancy when the circulating sex steroid concentrations increase very substantially; however, even during pregnancy, more than 99% of available binding sites on HSA remain unoccupied (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">11</a>). <em><span style="color: rgb(44, 130, 201)">It has long been hypothesized that HSA-bound testosterone may dissociate in the capillary bed of organs with long transit times, such as the liver and the brain, and may become biologically active (bioavailable) in these organs in addition to the unbound testosterone</span></em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">2</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">42</a>).</p><p></p><p><em><span style="color: rgb(44, 130, 201)">The HSA protein is encoded by a gene on chromosome 4 (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">43</a>), which contains 15 exons placed symmetrically in three domains that likely arose by triplication of a single ancestral gene. The HSA gene is translated into a 609−amino acid product from which a signal peptide and a propeptide are cleaved, yielding a 585−amino acid mature protein that is secreted into the circulation. </span></em>HSA in circulation can undergo nonenzymatic glycation by the formation of a Schiff base between <em>ε</em>-amino groups of lysine and arginine residues and glucose (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">44</a>). HSA is generally measured with dye-binding assays such as bromocresol green or bromocresol purple or with immunoassays (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">45</a>). The bromocresol green methods may overestimate HSA because of interference by acute-phase reactant proteins (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">46–48</a>), whereas the bromocresol purple method reportedly has high concordance with immunoassays (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">49</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">50</a>).</p><p></p><p><em><span style="color: rgb(44, 130, 201)">Major gaps remain in our understanding of the dynamics of free testosterone regulation by HSA.</span></em> Pardridge (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">42</a>) hypothesized that within the tissue capillaries, conformational changes in the HSA molecule caused by interactions between HSA and the endothelial wall could lead to an opening of the binding site coil and enhanced dissociation of testosterone from HSA. Indeed, the dissociation of testosterone from bovine serum albumin in the brain capillary is ∼50 times faster than dissociation from albumin <em>in vitro</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">42</a>). This increase in transportability of HSA-bound testosterone may result from interactions of HSA with specific receptors in the microcirculation; however, <em>in vivo</em> studies of HSA transport into the brain (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">51</a>) or liver (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">52</a>) microcirculation showed that the volume of distribution of HSA was no greater than in the vascular space. Others have postulated that the enhanced dissociation of testosterone from HSA in the capillaries results from the secretion of binding inhibitors from the endothelium (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">18</a>). Current models of the binding of testosterone to HSA are discussed further in a subsequent section.</p><p></p><p></p><p></p><p><span style="color: rgb(0, 0, 0)"><strong>Appraisal of the Prevailing Models of Testosterone Binding to Plasma Proteins</strong></span></p><p></p><p><span style="color: rgb(184, 49, 47)"><em><u>Most of the experimental data characterizing the association of testosterone with </u></em></span><u><span style="color: rgb(44, 130, 201)"><em>HSA </em></span></u><span style="color: rgb(184, 49, 47)"><em><u>and SHBG, which led to the conception of linear binding models of testosterone’s association with SHBG and HSA, including those by Vermeulen et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>), Södergard et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>), and Mazur (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">5</a>), were generated in the 1950s through the 1980s</u>.</em></span> <em><strong><span style="color: rgb(184, 49, 47)"><u>The resolution of the crystal structure of the liganded SHBG domains in the early 2000s was a major advance in our understanding of testosterone binding to SHBG. However, as we discuss subsequently, a paucity of experimental data supports the widely used assumptions of stoichiometry and the affinity of testosterone’s binding to SHBG </u></span></strong></em>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>).</p><p></p><p></p><p></p><p></p><p><strong>Table 1.</strong></p><p><strong><span style="color: rgb(184, 49, 47)">Factors Contributing to Erroneous Assumptions of Binding Affinity and Stoichiometry in Linear Models of Testosterone Binding to Its Cognate Binding Proteins</span></strong></p><p>[ATTACH=full]11398[/ATTACH]</p><p></p><table class='post-table ' style='width: 100%'><tr><th ><p>Contributing Factor</p></th></tr><tr><td ><p><span style="color: rgb(184, 49, 47)">1:1 Binding stoichiometry assumed without supporting experimental data</span></p></td></tr><tr><td ><p><span style="color: rgb(184, 49, 47)">Use of Scatchard plots to force a straight line through nonlinear experimental binding data</span></p></td></tr><tr><td ><p><span style="color: rgb(184, 49, 47)">Failure to account for alteration of binding equilibria during the separation of free and bound testosterone </span></p></td></tr><tr><td ><p><span style="color: rgb(184, 49, 47)">Variations in the estimates of binding affinity because of differences in the temperature at which binding isotherms and dialysis experiments were performed </span></p></td></tr><tr><td ><p><span style="color: rgb(184, 49, 47)">Variations in the estimates of binding affinity due to differences in dialysis conditions, including differences in the assay buffer composition and relative volumes of serum and assay buffers</span></p></td></tr><tr><td ><p><span style="color: rgb(184, 49, 47)"><strong><u>Limited ability to detect additional binding sites on </u></strong><u><strong>SHBG and </strong></u></span><u><span style="color: rgb(44, 130, 201)"><strong>HSA </strong></span></u><span style="color: rgb(184, 49, 47)"><strong><u>because of the narrow range of testosterone concentrations used in the binding experiments</u></strong></span></p></td></tr></table> </p><p></p><p><strong>Critical evaluation of the current model of testosterone binding to HSA</strong></p><p></p><p><span style="color: rgb(44, 130, 201)"><em>HSA consists of three domains [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 2(a</a>)] (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">60</a>); both domains II and III have a binding pocket formed mostly of hydrophobic and positively charged residues in which a variety of compounds bind (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">61</a>). It is widely believed that testosterone binds to HSA at a single site on domain IIA (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">62–64</a>) with a low-to-moderate affinity (i.e., an association constant of 2.0 to 4.1 × 104 L/mol at 37°C) and a fast dissociation half-time (∼1 second) </em></span>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">5</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">38</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">39</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">65</a>). <span style="color: rgb(44, 130, 201)"><em><u>As a result, all the equations for calculating free testosterone have used 1:1 stoichiometry of testosterone binding to HSA</u></em></span> [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 2(b</a>)] (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3–5</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">34</a>).</p><p></p><p><em><span style="color: rgb(44, 130, 201)"><u>In the years since these studies were published, limited experimental data in the literature have supported the commonly assumed 1:1 stoichiometry for the binding of testosterone to HSA</u> </span></em>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a><strong>)</strong> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">66</a>). For instance, experimental data published as early as 1954 by Eik-Nes <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">67</a>) suggested multiple, noninteracting testosterone binding sites on HSA. In 1978, Moll <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">68</a>) performed a detailed evaluation of the association of testosterone with HSA, and these authors also suspected multiple, identical, noninteracting testosterone binding sites on HSA. In 1982, Södergard <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>) conducted thermodynamic studies of the association of dihydrotestosterone with HSA and reported that the data pointed toward multiple binding sites on HSA. Ryan (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">69</a>) suggested the possibility of multiple binding sites for testosterone on HSA and a nonlinear binding relationship.<strong> <span style="color: rgb(44, 130, 201)"><em><u>The calculations of binding parameters based on the assumption of 1:1 stoichiometry may also be invalid</u></em></span> </strong>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">70</a>).<em><strong> <span style="color: rgb(44, 130, 201)"><u>Thus, although these trailblazers were suspicious of 1:1 stoichiometry, the methods and computational tools available to them were inherently limited in providing definitive evidence of stoichiometry, multiple binding sites with different binding affinities, or allostery in the binding of testosterone to HSA</u>. <u>Regardless, this same set of papers has been cited repeatedly over the years as the basis of the 1:1 stoichiometry for the binding of testosterone to HSA, although, in fact, these pioneering studies did not provide experimental data to support this assumption</u></span></strong></em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>).</p><p></p><p>As recently as the 1990s, Fischer <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">71</a>) concluded on the basis of studies that used equilibrium dialysis and circular dichroism that the second domain of the HSA molecule contained the primary binding site(s) for testosterone and acknowledged that “the data indicated the existence of cooperativity between secondary fatty acid-binding sites and the primary testosterone binding site.” Others also showed that for many ligands, the multiple binding sites on the HSA domains are allosterically coupled (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">72</a>). <em><strong><span style="color: rgb(44, 130, 201)"><u>It is conceivable that testosterone, like other ligands, may also have multiple binding sites with distinct affinities on HSA. Oversimplification of binding models and potentially erroneous assumptions can have major implications not only on estimates of testosterone’s bioavailability but also on putative competitive interactions with fatty acids and other hormones and drugs</u>.</span></strong></em></p><p><em></em></p><p><em><span style="color: rgb(44, 130, 201)">The dynamics of testosterone binding to HSA requires careful reexamination using modern experimental tools. <u><strong>Previous methods of comparing the solubility of testosterone in an aqueous buffer solution with its solubility in similarly buffered bovine serum albumin and using Scatchard analysis for equilibrium dialysis of testosterone with HSA were incapable of confirming multiple binding sites or identifying allosteric interactions between binding sites</strong></u><strong>. For instance, novel conformational probes that exhibit perturbations in their ground or excited-state optical properties in response to changes in their electronic environment can facilitate the characterization of the binding of hormones and drugs to HSA and evaluation of the competitive displacement by ligands. In addition, magnetic resonance spectroscopy using 13C-enriched probes can help map the spatial pockets of testosterone binding to HSA. </strong></span></em></p><p></p><p></p><p></p><p></p><p><strong>Methods for Determination of Free Testosterone</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)">Considering the high affinity of SHBG for testosterone binding, the SHBG-bound fraction is generally considered unavailable for biological action, and only the free and bioavailable testosterone fractions have been viewed as biologically active. </span></em>The need for accurate assessment of free testosterone levels in the diagnosis and treatment of hypogonadism has stimulated the development of a variety of methods (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 3</a>), which are discussed in detail in the following sections.</p><p></p><p></p><p></p><p></p><p><strong>Table 3.</strong></p><p><strong><span style="color: rgb(184, 49, 47)">The Relative Merits and Demerits of Various Methods of Measuring Free and Bioavailable Testosterone Levels</span></strong></p><p>[ATTACH=full]11400[/ATTACH]</p><p>[ATTACH=full]11401[/ATTACH]</p><table class='post-table ' style='width: 100%'><tr><th ><p>Bioavailable Testosterone</p></th></tr><tr><th ><p><strong>Method</strong></p></th><th ><p><strong>Merits</strong></p></th><th ><p><strong>Problems</strong></p></th></tr><tr><td ><p>Ammonium sulfate precipitation of SHBG-bound testosterone</p></td><td ><p>• Correlates well with free testosterone obtained by equilibrium dialysis</p></td><td ><p>• Technically difficult • Not easily automated • Few clinical laboratories measure it routinely •<strong> <span style="color: rgb(184, 49, 47)"><u>Conceptually measures non−SHBG-bound testosterone, </u></span><u><span style="color: rgb(44, 130, 201)">which approximates but does not equal HSA-bound </span></u><span style="color: rgb(184, 49, 47)"><u>plus unbound testosterone </u></span></strong></p></td></tr><tr><td ><p>Concanavalin A method</p></td><td ><p>• More selective and less variable than ammonium sulfate precipitation to precipitate SHBG</p></td><td ><p>• Technically difficult • Not easily automated • Not used currently by clinical laboratories •<u> <span style="color: rgb(184, 49, 47)"><strong>Measures non−SHBG-bound testosterone, </strong></span><span style="color: rgb(44, 130, 201)"><strong>which approximates but does not equal HSA-bound </strong></span></u><span style="color: rgb(184, 49, 47)"><u><strong>plus unbound testosterone</strong> </u></span></p></td></tr><tr><td ><p><strong><span style="color: rgb(184, 49, 47)">Calculated bioavailable testosterone</span></strong></p></td><td ><p><span style="color: rgb(184, 49, 47)"><strong>• Based on law-of-mass-action theory or empirical equations • Simple to obtain </strong></span></p></td><td ><p>• Correlation between different algorithms is poor unless revalidated in a local laboratory • <strong><span style="color: rgb(184, 49, 47)"><u>Dependent on correct estimation of the association constants for the binding of testosterone to SHBG (<em>K</em>T) and </u></span><u><span style="color: rgb(44, 130, 201)">HSA (<em>K</em>HSA)</span><span style="color: rgb(184, 49, 47)"> • Results affected by the quality of total testosterone and SHBG and </span><span style="color: rgb(44, 130, 201)">HSA </span></u><span style="color: rgb(184, 49, 47)"><u>measurements </u></span></strong></p></td></tr></table><table class='post-table ' style='width: 100%'><tr><th ><p>Free Testosterone</p></th></tr><tr><td ><p><strong><span style="color: rgb(184, 49, 47)">Equilibrium dialysis </span></strong></p></td><td ><p><strong><span style="color: rgb(184, 49, 47)">• The reference method against which other methods are compared</span></strong></p></td><td ><p><span style="color: rgb(184, 49, 47)"><strong>• <u>Technically difficult; operations in which the dialysis is performed vary across laboratories, contributing to high interlaboratory variability • Not easily automated • Few hospitals clinical laboratories perform this assay • Expensive • Relies on accuracy and precision of total testosterone</u></strong></span></p></td></tr><tr><td ><p>Ultracentrifugation</p></td><td ><p>• Comparable to equilibrium dialysis</p></td><td ><p>• Technically difficult • Not easily automated • Few clinical laboratories measure it routinely • Expensive • Relies on accuracy and precision of total testosterone</p></td></tr><tr><td ><p>Free androgen index</p></td><td ><p>• Represents the ratio of total testosterone/SHBG • Has been shown to correlate with free testosterone measurements • Simple to obtain</p></td><td ><p>• Overly simplistic and inaccurate measure of free testosterone concentrations • Poor indicator of gonadal status • Dependent on accurate measurements of total testosterone and SHBG • Most experts do not favor its use</p></td></tr><tr><td ><p>Analogue immunoassays</p></td><td ><p>• Commercially available kits • High throughput and precision • Has been shown to correlate with free testosterone measurements</p></td><td ><p>• Provides inaccurate estimates of free testosterone • Experts recommend against the use of direct analog assays for the measurement of free testosterone.</p></td></tr><tr><td ><p>Salivary testosterone</p></td><td ><p>• Simple to obtain</p></td><td ><p>• May not be an accurate marker of circulating free testosterone concentrations • Affected by sample desiccation, contamination by food and blood</p></td></tr><tr><td ><p><strong><span style="color: rgb(184, 49, 47)">Calculated free testosterone </span></strong></p></td><td ><p><strong><span style="color: rgb(184, 49, 47)">• Easy to use algorithms based on various models of testosterone binding to SHBG or empirical equations • Simple to obtain </span></strong></p></td><td ><p>• <strong><span style="color: rgb(184, 49, 47)"><u>Dependent upon correct estimates of the association constants and stoichiometry for binding of testosterone to SHBG and </u></span><span style="color: rgb(44, 130, 201)"><u>HSA</u></span><span style="color: rgb(184, 49, 47)"> • Accuracy and precision affected by the accuracy and precision of the total testosterone and SHBG assays </span></strong></p></td></tr></table> </p><p></p><p><strong>Equilibrium dialysis and its various embodiments</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)"><strong>Equilibrium dialysis is widely considered the reference method against which other methods are compared. It is technically demanding, and its performance is affected by assay conditions, which can result in high assay variability </strong></span></em>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">192</a>). Typically, the equilibrium dialysis procedure involves the dialysis of serum or plasma samples across a semipermeable cellulose membrane with a low-molecular-weight cutoff; protein-bound testosterone is retained, whereas free testosterone equilibrates across the dialysis membrane and can be measured in the dialysate either directly using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay or immunoassay or indirectly using a tracer. Indirect methods require adding a trace amount of radioactively labeled testosterone to the sample, and after equilibrium has been achieved, the proportion of tracer in the dialysate provides a measure of the percentage of free testosterone. Because free testosterone concentration can then be calculated by multiplying the percentage of the free fraction with the total testosterone concentration obtained from the same sample in a separate assay, accurate determination of total testosterone levels is necessary for accurate determination of free testosterone levels by this method.</p><p></p><p><em><span style="color: rgb(184, 49, 47)"><strong><u>Although a diligently conducted equilibrium dialysis assay accurately measures free testosterone levels, the method is fraught with operator-dependent errors. The protocol itself is labor-intensive, requiring repeated purification of the radioactive tracer, and is not readily amenable to high throughput</u>.</strong></span></em> Even some large commercial diagnostic laboratories have stopped offering this assay.<span style="color: rgb(184, 49, 47)"><em> <u><strong>Although equilibrium dialysis is widely considered to be the gold standard for measuring free testosterone, this method is subject to various sources of error that may contribute to inaccuracy and imprecision</strong></u><strong>. For instance, the dilution of serum or plasma may disturb the equilibrium between SHBG and its ligands (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">193</a>). Results may also be altered when solutes become attached to the dialysis apparatus or membrane or when there is an unequal distribution of free ligands between the two compartments as a result of</strong></em></span><strong> <span style="color: rgb(0, 0, 0)"><em>(1)</em></span><span style="color: rgb(44, 130, 201)"><em> inadequate time to reach equilibrium;</em></span><span style="color: rgb(0, 0, 0)"><em> (2)</em></span><span style="color: rgb(44, 130, 201)"><em> release of materials from the plate or membrane that interferes with the determination of concentration; and </em></span><span style="color: rgb(0, 0, 0)"><em>(3)</em></span></strong><span style="color: rgb(44, 130, 201)"><em><strong> the Donnan effect at low ionic strengths, which alters the distribution of charged particles near a semipermeable membrane so that they may not distribute evenly across the two sides of the membrane (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">194</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">195</a>). The ionic strength and pH of the dialysis buffer and the temperature at which dialysis is performed affect the equilibrium and the estimates of binding parameters. The batch-to-batch variability in adsorption characteristics of dialysis plates from different manufacturers may be an additional source of interassay variation. </strong></em></span>The Centers for Disease Control and Prevention’s (CDC’s) hormone standardization program is invested in improving clinical assays and minimizing factors that affect measurement variability (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">196</a>).</p><p></p><p></p><p><strong>Effects of temperature variations</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)">Steroid binding is affected by the temperature and may be 2.5 times higher at 4°C than at 37°C </span></em>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">9</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">197</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">198</a>). <em><span style="color: rgb(184, 49, 47)"><u>The seminal testosterone-binding experiments were performed at varying temperatures—some studies were performed with ice-cold ammonium sulfate (4°C) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">2</a>) or at 25°C (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">9</a>), which may affect binding equilibrium</u></span></em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>). For example, in a separate study characterizing temperature effects on cortisol protein binding by the equilibrium dialysis method, raising the temperature from 37°C to 41°C led to an increase of ∼80% in serum-free cortisol level (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">199</a>).</p><p></p><p><strong>Effects of assay buffer composition and buffer volumes</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)">The composition and ionic strength of the dialysis buffer affect the results of equilibrium dialysis experiments.</span></em> <em><span style="color: rgb(184, 49, 47)"><u>Experiments should ideally be performed using a dialysis buffer with an ionic composition that resembles that of human plasma, but this has not been the case in all studies. The assumption that the concentration of free ligands is equal on both sides of the membrane at equilibrium is not always valid</u></span></em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>). Most proteins have a charge and accumulate a set of neutralizing counterions. The Donnan effect, discussed previously, is a consequence of maintaining the overall electrical neutrality of the solution and may give spurious evidence of an association between a ligand and a protein of opposite charge when charged counterions are present in the buffer. <span style="color: rgb(184, 49, 47)"><u><em>Differences in the ratios of volumes of dialysis buffer to sample may also affect estimates of free testosterone; when the binding is nonlinear, the decrease in total analyte concentration can alter the free fraction</em></u></span> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>).</p><p></p><p><strong>Alteration of equilibria during physical separation of free and bound testosterone fractions</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)">Traditional assays for determining stoichiometry and association constants usually involve separation of bound and free forms of testosterone using equilibrium dialysis, ultracentrifugation, ammonium sulfate precipitation, or other chromatographic separation methods with a subsequent Scatchard plot of the ratio of bound testosterone to unbound testosterone [(bound/free testosterone); ordinate] plotted against the bound testosterone concentration [(bound); abscissa] </span></em>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">200</a>). <span style="color: rgb(184, 49, 47)"><u><em>The Scatchard analysis is a method of “linearizing” data from a saturation binding experiment to determine binding constants and estimates of the stoichiometry of the noninteracting sites. However, under several experimental conditions, the underlying assumptions in the Scatchard analysis are not met, and the use of the Scatchard analysis may yield inaccurate parameter estimation</em></u></span> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>).</p><p></p><p><strong><em><span style="color: rgb(184, 49, 47)"><u>Achieving standardization of dialysis conditions across laboratories has been difficult, resulting in substantial interlaboratory variations in reported results. Authors who measure free testosterone by equilibrium dialysis should provide details about their methodology to ensure reproducibility and interlaboratory comparability</u>.</span></em></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong>Computational Algorithms for Estimating Free and Bioavailable Testosterone Concentrations: Pitfalls and the Compelling Need for Accuracy in Calculated Free Testosterone</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)">Most hospital and commercial laboratories do not offer an equilibrium dialysis assay for free testosterone, most likely because of operational complexities in performing the assay and difficulties in automating the procedure; only a few academic and commercial laboratories offer this assay. Furthermore, efforts to standardize experimental conditions for the performance of equilibrium dialysis across the few commercial and academic laboratories that offer it have proven challenging.</span></em> Fortunately, LC-MS/MS methods for precise total testosterone measurements and high-sensitivity SHBG enzyme-linked immunosorbent assay are widely available. <span style="color: rgb(184, 49, 47)"><strong><em><u>Accordingly, an accurate algorithm, validated against the equilibrium dialysis measurement, can provide calculated free testosterone values with significantly higher precision and lower cost than can be achieved with equilibrium dialysis in many hospital laboratories</u>.</em></strong></span></p><p></p><p>Recognizing the practical difficulties that practicing clinicians face in obtaining precise and accurate measurements of free testosterone concentrations by the equilibrium dialysis method, an expert panel of the Endocrine Society concluded that “<em>calculated free testosterone, using high-quality testosterone and SHBG assays with well-defined reference intervals, is the most useful clinical marker</em>…” (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">205</a>). <em><strong><span style="color: rgb(184, 49, 47)">Accordingly, several groups have developed frameworks for computing free testosterone from SHBG, total testosterone, and HSA concentrations that can be broadly classified into three categories: </span><span style="color: rgb(0, 0, 0)">(1) </span><span style="color: rgb(184, 49, 47)">algorithms based on linear models of testosterone binding to SHBG, </span><span style="color: rgb(0, 0, 0)">(2)</span><span style="color: rgb(184, 49, 47)"> algorithms derived from empiric bootstrapping of data fits to mathematical forms, and </span><span style="color: rgb(0, 0, 0)">(3) </span><span style="color: rgb(184, 49, 47)"><u>algorithms based on nonlinear models incorporating allostery in the SHBG dimer</u>.</span></strong></em></p><p></p><p></p><p></p><p></p><p><strong>Calculated free testosterone based on linear models</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)"><u><strong>The algorithms published by Vermeulen et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>), Södergard et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>), and Mazer (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">5</a>) are all based on the linear model of testosterone binding to SHBG [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 3(a</a>)]. They all used Scatchard analysis to linearize data from a saturation-binding experiment to determine binding constants and estimates of the stoichiometry of noninteracting sites. However, under several experimental conditions, the underlying assumptions in the Scatchard analysis are not met, and the Scatchard analysis may yield inaccurate parameter estimation</strong></u><strong> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 1</a>)</strong>. For instance, the assumptions of linear regression, that the scatter of points about a line follows a normal (or Gaussian) distribution and the standard deviation is the same at every concentration of the analyte, are violated in a Scatchard plot, which alters the relationship between bound and free fractions. The use of the calculated values of the bound/free steroids further violates the assumption of linear regression that all uncertainty is in the Y variable, whereas the X variable is known with complete certainty.</span></em> <em><span style="color: rgb(184, 49, 47)"><u><strong>Because of the nonlinear nature of binding and allosteric interactions between binding sites, the linear transformation of the binding data to force a straight line through nonlinear data renders these historical estimates of binding affinity and capacity prone to error</strong></u>. Nonlinear computational tools may be more suitable for binding events, which involve allosteric interactions and are nonlinear; however, these methods have not been used in the literature for the analyses of data related to testosterone binding to SHBG or HSA.</span></em></p><p><em></em></p><p><em><strong><span style="color: rgb(184, 49, 47)"><u>These algorithms use different association constants of testosterone binding to </u></span><u><span style="color: rgb(44, 130, 201)">HSA</span></u><span style="color: rgb(184, 49, 47)"><u> and SHBG and therefore yield slightly different estimates of free testosterone</u>. For example, the Vermeulen et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>) equation used association constants of </span><span style="color: rgb(44, 130, 201)">3.6 × 104 </span><span style="color: rgb(184, 49, 47)">and 1 × 109 L/mol for </span><span style="color: rgb(44, 130, 201)">HSA</span><span style="color: rgb(184, 49, 47)"> and SHBG, respectively, whereas the Södergard et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>) algorithm used </span><span style="color: rgb(44, 130, 201)">4.06 × 104</span><span style="color: rgb(184, 49, 47)"> and 5.97 × 108 L/ mol, respectively. All three equations, especially the Vermeulen et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>) equation, have been widely used in the literature and in commercial laboratories. <u>The calculated free testosterone values derived using these equations correlated with free testosterone concentrations measured by equilibrium dialysis in some studies (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">214</a>) but displayed substantial systematic differences from values derived using the equilibrium dialysis method and from each othe</u></span></strong></em><span style="color: rgb(184, 49, 47)"><u>r </u></span>(<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">6</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">34</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">215</a>–<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">218</a>).</p><p></p><p>Hackbarth <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">218</a>) evaluated five separate equations in two patient groups with different sex distributions. They defined percentage differences above 20% to be unacceptable; depending on the equation, 32% to 72% of males and 29% to 57% of females displayed an unacceptable agreement between levels of calculated free testosterone and measured free testosterone by equilibrium dialysis. In addition, 14.9% of males and 11.1% of females showed poor fit by all five equations.</p><p></p><p></p><p></p><p></p><p><strong>Calculated free testosterone from empiric and bootstrap fitting approaches</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)">Ly and Handelsman (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">6</a>) analyzed a data set comprising >4000 blood samples in which free and total testosterone and SHBG concentrations were measured; dividing the data set into samples with serum total testosterone above and below 5 nM, they used a bootstrap regression modeling approach free of assumptions about theoretical binding equilibria to develop an empirical equation for free testosterone in terms of total testosterone and SHBG. Later, Sartorius et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">217</a>) created a variety of formulas for evaluation by bootstrap resampling to identify the best-fit model according to entropy reduction and improve upon the previous empirical calculated free testosterone equation.</span></em> <strong><em><span style="color: rgb(184, 49, 47)"><u>This algorithm, like the others, is highly dependent on the accuracy and precision of the total testosterone and SHBG assays, which affects the accuracy and precision of calculated free testosterone (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">219</a>). Furthermore, regression equations derived empirically in one patient population may not necessarily apply to another population, especially to a population with substantially different SHBG concentrations. In a different patient population, there is no reason to believe that best-fit parameters will be the same as in the test population. </u></span><span style="color: rgb(44, 130, 201)"><u>In addition, these methods do not have a testable binding model that can be subjected to experimental validation or improved upon to incorporate other variables or new knowledge of the dynamics of testosterone binding to its cognate binding proteins or for personalization to specific conditions or disease states</u>.</span></em></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong>Calculated free testosterone using an algorithm that incorporates experimentally observed nonlinear binding dynamics and allosteric interaction between binding sites</strong></p><p></p><p>We recently investigated the source of systematic discrepancies between free testosterone values computed using the simple linear model, which formed the basis of the Vermeulen <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>), Södergard <em>et al.</em> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>), and Mazer (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">5</a>) equations, and free testosterone measured using equilibrium dialysis. <em><span style="color: rgb(184, 49, 47)"><strong><u>These discrepancies between free testosterone calculated using these linear binding models and free testosterone measured using equilibrium dialysis are most likely the result of the erroneous assumptions of the dynamics of testosterone binding to SHBG</u>. Recent studies of testosterone binding to SHBG using modern biophysical techniques suggest that SHBG circulates as a homodimer and that there is complex allosteric interaction between the two binding sites on the SHBG dimer, <u>such that the binding affinities of the two sites are not identical (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">34</a>). The computational algorithm based on this novel multistep ensemble allosteric model (EAM)</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">34</a>) of testosterone binding to SHBG provided estimates of free testosterone levels that closely matched free testosterone levels measured using the equilibrium dialysis method in samples derived from men and women in two randomized clinical trials (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">220</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">221</a>).</strong></span></em> <em><strong><span style="color: rgb(184, 49, 47)"><u>The calculated free testosterone level obtained using the prevailing linear model was systematically lower than those measured by equilibrium dialysis. In <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 4</a>, we show that calculated free testosterone values across age deciles in the Framingham Heart Study computed by the linear model are lower than those computed by the allosteric model.</u></span><span style="color: rgb(44, 130, 201)"><u> The EAM model is based on experimentally derived binding affinity and dynamics, which can be verified experimentally and improved upon with additional information about other variables that determine free testosterone concentrations</u>.</span></strong></em></p><p></p><p></p><p></p><p></p><p></p><p></p><p><strong>Lack of Standardization of Free Testosterone Measurement Methods and Unavailability of Harmonized Reference Ranges for Free Testosterone</strong></p><p></p><p><em><span style="color: rgb(184, 49, 47)">Le et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">222</a>) surveyed 120 academic and community laboratories in the United States to characterize the distribution of assays and the associated reference values for free testosterone. In all, 84% of the surveyed laboratories sent their samples for free testosterone measurement to larger centralized reference laboratories (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">222</a>). These large commercial laboratories offered a variety of methods, including ultracentrifugation, radioimmunoassay, and calculation-based algorithms, as well as equilibrium dialysis (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">222</a>). Many clinical laboratories used calculated free testosterone based on published linear equations (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3</a>).</span><strong> <span style="color: rgb(184, 49, 47)"><u>The laboratories reported wide variations in the reference ranges. </u></span><span style="color: rgb(44, 130, 201)"><u>Only 30 of the laboratories surveyed would confirm that validation studies had been performed,</u></span><span style="color: rgb(184, 49, 47)"><u> and the authors advised that reference ranges provided by manufacturers and laboratories should be interpreted with caution</u>.</span></strong></em></p><p><em><strong><span style="color: rgb(184, 49, 47)"></span></strong></em></p><p><em><strong><span style="color: rgb(184, 49, 47)"><u>In a survey of 12 academic laboratories, 12 community medical laboratories, and one national laboratory, Lazarou et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">223</a>) found 17 and 13 different sets of reference values for total and free testosterone, respectively, which were established largely without clinical considerations</u>.</span> <span style="color: rgb(44, 130, 201)">Recently, Bhasin et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">224</a>) reported reference ranges for calculated free testosterone concentrations in a large, rigorously collected sample of community-dwelling men. In healthy young men of the Framingham Heart Study who were 19 to 40 years of age, the lower limit of the normal range, defined as the 2.5th percentile of calculated free testosterone, was 70 pg/mL (242.7 pmol/L) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">198</a>).</span></strong></em></p><p></p><p></p><p></p><p></p><p></p><p></p><p><strong>Clinical Implications and Recommendations</strong></p><p></p><p>Male hypogonadism is a clinical condition characterized by the presence of typical signs and symptoms in the setting of consistently low serum testosterone concentrations.<em><strong> <span style="color: rgb(184, 49, 47)"><u>The Endocrine Society guidelines currently suggest measuring free testosterone levels in men in whom total testosterone concentrations are near the lower limit of the normal range and in men with conditions that affect SHBG concentrations and render total testosterone a less reliable index of gonadal function</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">206</a>). If the free hormone hypothesis is correct, free testosterone should serve as the benchmark for biochemical confirmation of hypogonadism. </span><span style="color: rgb(44, 130, 201)"><u>Accurate determination of free testosterone values is therefore central to an accurate diagnosis of hypogonadism</u></span><span style="color: rgb(184, 49, 47)">.</span></strong></em></p><p></p><p><span style="color: rgb(26, 188, 156)"><strong><em><u>The direct analog assays for free testosterone determination are inaccurate and should not be used.</u> </em></strong></span><em><strong><span style="color: rgb(26, 188, 156)">However, a confluence of factors related to the regulatory process, economic considerations, and difficulties in performing equilibrium dialysis methods in many hospital laboratories has led to their surprising endurance despite their known inaccuracy.</span></strong></em> Historically, laboratory-certifying bodies, such as the Clinical Laboratory Improvement Amendments, have certified laboratories and assays mostly on the basis of process measures; unlike the CDC and its Hormone Assay Standardization program for testosterone, these bodies have generally not required accuracy-based benchmarks. Similarly, the requirement in the assay approval process for demonstration of comparability to a previously approved assay enables new tracer analog assays to be approved because they can demonstrate comparability to previously approved analog methods.</p><p></p><p><strong><em><span style="color: rgb(184, 49, 47)">Equilibrium dialysis is the reference method for free testosterone determination, but this assay is not always available to clinicians in all hospital laboratories; in addition, t<u>here are substantial interlaboratory variations because of the lack of standardization of assay conditions, making it difficult for practicing endocrinologists to interpret free testosterone levels</u>. </span></em></strong><span style="color: rgb(44, 130, 201)"><em><strong><u>Mechanisms to harmonize the equilibrium dialysis procedure across laboratories are needed. Until equilibrium dialysis methods can be standardized across laboratories, a computational framework that accurately captures the dynamics of testosterone to SHBG and HSA interactions in calculating free testosterone values is an unmet need for precise clinical diagnosis</u>. The EAM appears to be an accurate and testable model for calculating free testosterone levels, but this model needs further validation in large populations.</strong></em></span></p></blockquote><p></p>
[QUOTE="madman, post: 190020, member: 13851"] Much more going on here than SHBG:T binding when it comes to cFT! Patiently waiting on the completion of Phase II for the TruT (cFTZ) Algorithm [B]R44G045011 [COLOR=rgb(184, 49, 47)](JASUJA, RAVI)[/COLOR] NIH/NIA Phase II: [COLOR=rgb(184, 49, 47)]Research and Commercialization of TruT Algorithm[/COLOR] Role: Principal Investigator Sep 15, 2017 - [COLOR=rgb(184, 49, 47)]May 31, 2021[/COLOR][/B] [ATTACH type="full" alt="Screenshot (2260).png"]11404[/ATTACH] [B]A Reappraisal of Testosterone's Binding in Circulation: [COLOR=rgb(184, 49, 47)]Physiological and Clinical Implications [/COLOR](2017)[/B] [COLOR=rgb(44, 130, 201)]Anna L Goldman,[/COLOR][COLOR=rgb(184, 49, 47)] [B]Shalender Bhasin[/B][/COLOR][COLOR=rgb(44, 130, 201)], Frederick C W Wu, Meenakshi Krishna, Alvin M Matsumoto, [/COLOR][COLOR=rgb(184, 49, 47)][B]Ravi Jasuja[/B][/COLOR] [B]Abstract[/B] [I][COLOR=rgb(184, 49, 47)][U]In the circulation, testosterone and other sex hormones are bound to binding proteins, which play an important role in regulating their transport, distribution, metabolism, and biological activity[/U].[/COLOR][/I] According to the free hormone hypothesis, which has been debated extensively, only the unbound or free fraction is biologically active in target tissues. [I][COLOR=rgb(184, 49, 47)]Consequently,[U] accurate determination of the partitioning of testosterone between bound and free fractions is central to our understanding of how it's delivery to the target tissues and biological activity are regulated[/U] and consequently to the diagnosis and treatment of androgen disorders in men and women.[/COLOR][/I] [B][I][COLOR=rgb(184, 49, 47)]Here, we present a historical perspective on the evolution of our understanding of the binding of testosterone to circulating binding proteins. [U]On the basis of an appraisal of the literature as well as experimental data, we show that the assumptions of stoichiometry, binding dynamics, and the affinity of the prevailing models of testosterone binding to sex hormone-binding globulin and [/U][/COLOR][COLOR=rgb(44, 130, 201)][U]human serum albumin[/U][/COLOR][COLOR=rgb(184, 49, 47)][U] are not supported by published experimental data and are most likely inaccurate[/U].[/COLOR] [/I][/B]This review offers some guiding principles for the application of free testosterone measurements in the diagnosis and treatment of patients with androgen disorders. The growing number of testosterone prescriptions and widely recognized problems with the direct measurement as well as the computation of free testosterone concentrations render this critical review timely and clinically relevant. [B]Essential Points[/B] [COLOR=rgb(184, 49, 47)]Most circulating testosterone is bound to its cognate binding proteins—sex hormone−binding globulin (SHBG), [/COLOR][COLOR=rgb(44, 130, 201)][U]human serum albumin (HSA)[/U][/COLOR][COLOR=rgb(184, 49, 47)], cortisol-binding globulin, and orosomucoid; [U]these binding proteins play an important role in regulating the transport, tissue delivery, bioactivity, and metabolism of testosterone[/U] The physiochemical characteristics and dynamics of the binding of testosterone to its binding proteins are poorly understood; [U]oversimplified assumptions of stoichiometry, binding dynamics, and binding affinity have contributed to the development of inaccurate linear binding models of testosterone to SHBG and [/U][/COLOR][COLOR=rgb(44, 130, 201)][U]HSA[/U] [/COLOR] [COLOR=rgb(184, 49, 47)][U]The ensemble allosteric model of the binding of testosterone to SHBG developed from recent studies using modern biophysical techniques suggests that testosterone binding to SHBG is a complex, multistep process that involves interbinding site allostery [/U][/COLOR] [COLOR=rgb(44, 130, 201)][U]The dynamics of the binding of testosterone to HSA[/U][/COLOR][COLOR=rgb(184, 49, 47)][U], orosomucoid, and corticosteroid-binding globulin also require careful reexamination because the roles of these binding proteins in regulating circulating testosterone concentrations remain incompletely understood[/U] I[U]f the free hormone hypothesis is correct ([I]i.e.,[/I] only free testosterone is biologically active), accurate determination and harmonized reference ranges for free testosterone are necessary to diagnose androgen disorders in men and women Methods for the measurement of free testosterone levels are fraught with potential problems, including poor precision, inaccuracy, and low specificity, and reliable assays are not readily available to practicing clinicians; therefore, [/U][/COLOR][COLOR=rgb(44, 130, 201)][U]algorithms based on valid binding models that can be used to estimate circulating free testosterone levels[/U][/COLOR][COLOR=rgb(184, 49, 47)][U] are needed to facilitate sound clinical decision making[/U] [I][U]Binding proteins in the peripheral circulation are important in regulating the transport, bioavailability, and metabolism of their cognate ligands, such as steroid hormones, fatty acids, vitamins, and drugs[/U]. The major sex steroid hormones—testosterone, 5α-dihydrotestosterone, and 17β-estradiol—bind predominantly to sex hormone−binding globulin (SHBG) and to [/I][/COLOR][COLOR=rgb(44, 130, 201)][I]human serum albumin (HSA)[/I][/COLOR][COLOR=rgb(184, 49, 47)][I] and to a lesser extent to corticosteroid-binding globulin (CBG) and orosomucoid. SHBG, which is secreted by the liver, binds to testosterone with high affinity and is an important determinant of the distribution of circulating testosterone into its bound and free fractions ([URL='https://www.excelmale.com/forum/javascript%3A;']1[/URL]). [/I][/COLOR][COLOR=rgb(44, 130, 201)][I][U]HSA is one of the most abundant and versatile proteins in circulation; although it binds testosterone with lower affinity than SHBG does, its high binding capacity and high concentration allow it to buffer fluctuations in testosterone levels[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']1[/URL]).[/I][/COLOR] The characteristics of testosterone binding to CBG and orosomucoid and the biological roles of these binding proteins in regulating testosterone bioavailability remain incompletely understood. [I][B]Total testosterone[/B][/I] [I][COLOR=rgb(184, 49, 47)]refers to the sum of the concentrations of protein-bound and unbound testosterone in circulation. The fraction of circulating testosterone that is unbound to any plasma protein is referred to as the[/COLOR][/I] [I][COLOR=rgb(0, 0, 0)][B]free testosterone fraction[/B][/COLOR][/I][B]. [/B][COLOR=rgb(184, 49, 47)][I]The term[/I][/COLOR] [I][B]bioavailable testosterone[/B][/I] [COLOR=rgb(184, 49, 47)][I]refers to the fraction of circulating testosterone that is not bound to SHBG and largely represents the sum of free testosterone plus [/I][/COLOR][COLOR=rgb(44, 130, 201)][I]HSA-bound testosterone[/I][/COLOR] ([URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 1[/URL]) ([URL='https://www.excelmale.com/forum/javascript%3A;']2[/URL]); [COLOR=rgb(44, 130, 201)][I][U]the term reflects the view that HSA-bound testosterone, which is bound with low affinity, can dissociate from HSA in the tissue capillaries and effectively be available for biological activity[/U].[/I][/COLOR] [I][COLOR=rgb(184, 49, 47)]The free testosterone fraction can be measured directly by the equilibrium dialysis or ultrafiltration method or calculated from total testosterone, SHBG, and [/COLOR][COLOR=rgb(44, 130, 201)]HSA [/COLOR][COLOR=rgb(184, 49, 47)]concentrations using published mass action binding algorithms[/COLOR][/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']3–6[/URL]). The bioavailable fraction can be measured using the ammonium sulfate precipitation method or the concanavalin A method, or it can be calculated from total testosterone, SHBG, and HSA concentrations ([URL='https://www.excelmale.com/forum/javascript%3A;']7[/URL]). Although the pioneers who originated the concept of bioavailable testosterone envisioned it as the sum of HSA-bound and unbound fractions of circulating testosterone ([URL='https://www.excelmale.com/forum/javascript%3A;']2[/URL]), the methods used to measure bioavailable testosterone concentrations, namely, the ammonium sulfate precipitation and concanavalin A methods, quantitate it as the non−SHBG-bound fraction of circulating testosterone, which approximates but is not equivalent to its original conceptualization as the sum of HSA-bound plus unbound testosterone levels ([URL='https://www.excelmale.com/forum/javascript%3A;']8[/URL]). [ATTACH type="full" alt="Screenshot (2378).png"]11395[/ATTACH] [B][COLOR=rgb(184, 49, 47)]Partitioning of testosterone in the systemic circulation.[/COLOR] Circulating testosterone is bound tightly to SHBG[COLOR=rgb(26, 188, 156)] (green = high-affinity binding) [/COLOR]and [COLOR=rgb(0, 0, 0)]weakly to[/COLOR][COLOR=rgb(44, 130, 201)] albumin, [/COLOR][COLOR=rgb(0, 0, 0)]orosomucoid (ORM), and CBG [/COLOR][COLOR=rgb(44, 130, 201)](blue = low-affinity binding)[/COLOR] ([URL='https://www.excelmale.com/forum/javascript%3A;']11[/URL]). [COLOR=rgb(184, 49, 47)][U]Only 1% to 4% of circulating testosterone is unbound or free[/U].[/COLOR] The combination of free and [COLOR=rgb(44, 130, 201)]albumin-bound testosterone[/COLOR] is also referred to as the “bioavailable testosterone” fraction. [/B] [COLOR=rgb(184, 49, 47)][I][U]The validity of calculated bioavailable and free testosterone is predicated on the accuracy of binding protein and testosterone concentrations and on the veracity of the assumptions of the association stoichiometry, binding affinities, and binding dynamics underlying the molecular binding model[/U].[/I][/COLOR] The foundational assumptions about the relationship between testosterone and its binding proteins and estimates of the biophysical parameters of testosterone binding to its cognate binding proteins, upon which many extant algorithms for computing free testosterone are based, have undergone recent reappraisal and are discussed later. [COLOR=rgb(184, 49, 47)][I][U]Data from the experimental studies performed in the 1960s and 1970s have been extrapolated without acknowledgment of the lack of experimental support for the underlying assumptions about linearity ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']9–11[/URL]) or of the methodological limitations described by the original authors[/U]. [U]Collectively, these have led to an oversimplification of binding models based on somewhat erroneous assumptions of stoichiometry, binding affinity, and binding dynamics[/U]. [/I][/COLOR] The rapid growth of testosterone prescriptions during the last decade ([URL='https://www.excelmale.com/forum/javascript%3A;']12[/URL]) has refocused attention on the critical need for accurate determination of free testosterone in the diagnostic evaluation of men with a suspected androgen deficiency and for rational dosing and monitoring of testosterone replacement therapy. [I][COLOR=rgb(184, 49, 47)]Accordingly, an expository review of the published data and prevailing models of testosterone binding is timely. [U]Here, we present a historical perspective of the evolution of our understanding of the binding and bioavailability of testosterone. This review attempts to provide a comprehensive and critical appraisal of the prevailing models of testosterone binding to SHBG and[/U][/COLOR][COLOR=rgb(44, 130, 201)][U] HSA[/U][/COLOR][COLOR=rgb(184, 49, 47)][U], the associated biophysical parameters, and their underlying assumptions and limitations[/U].[/COLOR][/I] [I][COLOR=rgb(184, 49, 47)][U]We discuss how recent advances in the computational and biophysical techniques have begun to unravel the multistep dynamics of testosterone binding to its cognate binding proteins, including the allosteric interactions between the testosterone binding sites on the SHBG dimer[/U]. [/COLOR][/I]This review also provides a contemporary perspective on the validity of the free hormone hypothesis and the clinical implications of these findings in the diagnosis, treatment, and monitoring of men with hypogonadism. [B]Biology of Binding Proteins and Their Role in the Transport, Distribution, Metabolism, and Bioavailability of Testosterone[/B] [COLOR=rgb(184, 49, 47)][I]At least four structurally distinct binding proteins are known to bind testosterone in human circulation: SHBG, [/I][/COLOR][COLOR=rgb(44, 130, 201)][I]HSA[/I][/COLOR][COLOR=rgb(184, 49, 47)][I], CBG, and orosomucoid. [/I][/COLOR]Among these, SHBG has received the most attention because of its high binding affinity for testosterone. [I][COLOR=rgb(184, 49, 47)]T[U]hese binding proteins influence the tissue bioavailability and metabolic clearance rate of testosterone by regulating the amount of free testosterone available for biological action in the tissue[/U]. The roles of[/COLOR][COLOR=rgb(44, 130, 201)] HSA[/COLOR][COLOR=rgb(184, 49, 47)], CBG, and orosomucoid in regulating testosterone’s bioavailability are less well understood, [U]and we do not know how disease states or conditions that may differentially alter the circulating concentrations of [/U][/COLOR][U][COLOR=rgb(44, 130, 201)]HSA[/COLOR][/U][COLOR=rgb(184, 49, 47)][U], CBG, and orosomucoid impact the binding of testosterone to SHBG[/U].[/COLOR][/I] [I][COLOR=rgb(184, 49, 47)][B][U]Current computations of free and bioavailable testosterone account only for the potential impact of alterations in [/U][/B][/COLOR][U][COLOR=rgb(44, 130, 201)][B]HSA[/B][/COLOR][/U][COLOR=rgb(184, 49, 47)][B][U] and SHBG, ignoring CBG and orosomucoid and other potentially interacting proteins and steroid hormones[/U][/B].[/COLOR][/I] [B]HSA[/B] [I][COLOR=rgb(44, 130, 201)][U]HSA is the most abundant protein in the human circulation, accounting for 60% of the total serum protein content and having a concentration of 30 to 50 g/L (450 to 750 µM) ([URL='https://www.excelmale.com/forum/javascript%3A;']35[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']36[/URL]). From 33% to 54% of testosterone binds with low affinity to HSA, with an association constant of 2.0 to 4.1 × 104 L/mol at 37°C[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']5[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']37–39[/URL]). [/COLOR][/I]Albumin Catania (580 Lys-Leu-Pro-COOH) ([URL='https://www.excelmale.com/forum/javascript%3A;']40[/URL]) and albumin Roma (321 Glu-Lys) ([URL='https://www.excelmale.com/forum/javascript%3A;']41[/URL]) are known variants that impact the affinity of HSA for testosterone; albumin Roma has a decreased affinity for testosterone, and it is unknown if albumin Catania has an increased or decreased affinity. The high capacity of HSA for binding steroids is particularly highlighted during pregnancy when the circulating sex steroid concentrations increase very substantially; however, even during pregnancy, more than 99% of available binding sites on HSA remain unoccupied ([URL='https://www.excelmale.com/forum/javascript%3A;']11[/URL]). [I][COLOR=rgb(44, 130, 201)]It has long been hypothesized that HSA-bound testosterone may dissociate in the capillary bed of organs with long transit times, such as the liver and the brain, and may become biologically active (bioavailable) in these organs in addition to the unbound testosterone[/COLOR][/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']2[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']42[/URL]). [I][COLOR=rgb(44, 130, 201)]The HSA protein is encoded by a gene on chromosome 4 ([URL='https://www.excelmale.com/forum/javascript%3A;']43[/URL]), which contains 15 exons placed symmetrically in three domains that likely arose by triplication of a single ancestral gene. The HSA gene is translated into a 609−amino acid product from which a signal peptide and a propeptide are cleaved, yielding a 585−amino acid mature protein that is secreted into the circulation. [/COLOR][/I]HSA in circulation can undergo nonenzymatic glycation by the formation of a Schiff base between [I]ε[/I]-amino groups of lysine and arginine residues and glucose ([URL='https://www.excelmale.com/forum/javascript%3A;']44[/URL]). HSA is generally measured with dye-binding assays such as bromocresol green or bromocresol purple or with immunoassays ([URL='https://www.excelmale.com/forum/javascript%3A;']45[/URL]). The bromocresol green methods may overestimate HSA because of interference by acute-phase reactant proteins ([URL='https://www.excelmale.com/forum/javascript%3A;']46–48[/URL]), whereas the bromocresol purple method reportedly has high concordance with immunoassays ([URL='https://www.excelmale.com/forum/javascript%3A;']49[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']50[/URL]). [I][COLOR=rgb(44, 130, 201)]Major gaps remain in our understanding of the dynamics of free testosterone regulation by HSA.[/COLOR][/I] Pardridge ([URL='https://www.excelmale.com/forum/javascript%3A;']42[/URL]) hypothesized that within the tissue capillaries, conformational changes in the HSA molecule caused by interactions between HSA and the endothelial wall could lead to an opening of the binding site coil and enhanced dissociation of testosterone from HSA. Indeed, the dissociation of testosterone from bovine serum albumin in the brain capillary is ∼50 times faster than dissociation from albumin [I]in vitro[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']42[/URL]). This increase in transportability of HSA-bound testosterone may result from interactions of HSA with specific receptors in the microcirculation; however, [I]in vivo[/I] studies of HSA transport into the brain ([URL='https://www.excelmale.com/forum/javascript%3A;']51[/URL]) or liver ([URL='https://www.excelmale.com/forum/javascript%3A;']52[/URL]) microcirculation showed that the volume of distribution of HSA was no greater than in the vascular space. Others have postulated that the enhanced dissociation of testosterone from HSA in the capillaries results from the secretion of binding inhibitors from the endothelium ([URL='https://www.excelmale.com/forum/javascript%3A;']18[/URL]). Current models of the binding of testosterone to HSA are discussed further in a subsequent section. [COLOR=rgb(0, 0, 0)][B]Appraisal of the Prevailing Models of Testosterone Binding to Plasma Proteins[/B][/COLOR] [COLOR=rgb(184, 49, 47)][I][U]Most of the experimental data characterizing the association of testosterone with [/U][/I][/COLOR][U][COLOR=rgb(44, 130, 201)][I]HSA [/I][/COLOR][/U][COLOR=rgb(184, 49, 47)][I][U]and SHBG, which led to the conception of linear binding models of testosterone’s association with SHBG and HSA, including those by Vermeulen et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL]), Södergard et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL]), and Mazur ([URL='https://www.excelmale.com/forum/javascript%3A;']5[/URL]), were generated in the 1950s through the 1980s[/U].[/I][/COLOR] [I][B][COLOR=rgb(184, 49, 47)][U]The resolution of the crystal structure of the liganded SHBG domains in the early 2000s was a major advance in our understanding of testosterone binding to SHBG. However, as we discuss subsequently, a paucity of experimental data supports the widely used assumptions of stoichiometry and the affinity of testosterone’s binding to SHBG [/U][/COLOR][/B][/I]([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL]). [B]Table 1. [COLOR=rgb(184, 49, 47)]Factors Contributing to Erroneous Assumptions of Binding Affinity and Stoichiometry in Linear Models of Testosterone Binding to Its Cognate Binding Proteins[/COLOR][/B] [ATTACH type="full" alt="Screenshot (2379).png"]11398[/ATTACH] [TABLE][TR][TH]Contributing Factor[/TH][/TR] [TR][TD] [COLOR=rgb(184, 49, 47)]1:1 Binding stoichiometry assumed without supporting experimental data[/COLOR] [/TD][/TR] [TR][TD] [COLOR=rgb(184, 49, 47)]Use of Scatchard plots to force a straight line through nonlinear experimental binding data[/COLOR] [/TD][/TR] [TR][TD] [COLOR=rgb(184, 49, 47)]Failure to account for alteration of binding equilibria during the separation of free and bound testosterone [/COLOR] [/TD][/TR] [TR][TD] [COLOR=rgb(184, 49, 47)]Variations in the estimates of binding affinity because of differences in the temperature at which binding isotherms and dialysis experiments were performed [/COLOR] [/TD][/TR] [TR][TD] [COLOR=rgb(184, 49, 47)]Variations in the estimates of binding affinity due to differences in dialysis conditions, including differences in the assay buffer composition and relative volumes of serum and assay buffers[/COLOR] [/TD][/TR] [TR][TD] [COLOR=rgb(184, 49, 47)][B][U]Limited ability to detect additional binding sites on [/U][/B][U][B]SHBG and [/B][/U][/COLOR][U][COLOR=rgb(44, 130, 201)][B]HSA [/B][/COLOR][/U][COLOR=rgb(184, 49, 47)][B][U]because of the narrow range of testosterone concentrations used in the binding experiments[/U][/B][/COLOR] [/TD][/TR][/TABLE] [B]Critical evaluation of the current model of testosterone binding to HSA[/B] [COLOR=rgb(44, 130, 201)][I]HSA consists of three domains [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 2(a[/URL])] ([URL='https://www.excelmale.com/forum/javascript%3A;']60[/URL]); both domains II and III have a binding pocket formed mostly of hydrophobic and positively charged residues in which a variety of compounds bind ([URL='https://www.excelmale.com/forum/javascript%3A;']61[/URL]). It is widely believed that testosterone binds to HSA at a single site on domain IIA ([URL='https://www.excelmale.com/forum/javascript%3A;']62–64[/URL]) with a low-to-moderate affinity (i.e., an association constant of 2.0 to 4.1 × 104 L/mol at 37°C) and a fast dissociation half-time (∼1 second) [/I][/COLOR]([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']5[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']38[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']39[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']65[/URL]). [COLOR=rgb(44, 130, 201)][I][U]As a result, all the equations for calculating free testosterone have used 1:1 stoichiometry of testosterone binding to HSA[/U][/I][/COLOR] [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 2(b[/URL])] ([URL='https://www.excelmale.com/forum/javascript%3A;']3–5[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']34[/URL]). [I][COLOR=rgb(44, 130, 201)][U]In the years since these studies were published, limited experimental data in the literature have supported the commonly assumed 1:1 stoichiometry for the binding of testosterone to HSA[/U] [/COLOR][/I]([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL][B])[/B] ([URL='https://www.excelmale.com/forum/javascript%3A;']66[/URL]). For instance, experimental data published as early as 1954 by Eik-Nes [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']67[/URL]) suggested multiple, noninteracting testosterone binding sites on HSA. In 1978, Moll [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']68[/URL]) performed a detailed evaluation of the association of testosterone with HSA, and these authors also suspected multiple, identical, noninteracting testosterone binding sites on HSA. In 1982, Södergard [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL]) conducted thermodynamic studies of the association of dihydrotestosterone with HSA and reported that the data pointed toward multiple binding sites on HSA. Ryan ([URL='https://www.excelmale.com/forum/javascript%3A;']69[/URL]) suggested the possibility of multiple binding sites for testosterone on HSA and a nonlinear binding relationship.[B] [COLOR=rgb(44, 130, 201)][I][U]The calculations of binding parameters based on the assumption of 1:1 stoichiometry may also be invalid[/U][/I][/COLOR] [/B]([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL]) ([URL='https://www.excelmale.com/forum/javascript%3A;']70[/URL]).[I][B] [COLOR=rgb(44, 130, 201)][U]Thus, although these trailblazers were suspicious of 1:1 stoichiometry, the methods and computational tools available to them were inherently limited in providing definitive evidence of stoichiometry, multiple binding sites with different binding affinities, or allostery in the binding of testosterone to HSA[/U]. [U]Regardless, this same set of papers has been cited repeatedly over the years as the basis of the 1:1 stoichiometry for the binding of testosterone to HSA, although, in fact, these pioneering studies did not provide experimental data to support this assumption[/U][/COLOR][/B][/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL]). As recently as the 1990s, Fischer [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']71[/URL]) concluded on the basis of studies that used equilibrium dialysis and circular dichroism that the second domain of the HSA molecule contained the primary binding site(s) for testosterone and acknowledged that “the data indicated the existence of cooperativity between secondary fatty acid-binding sites and the primary testosterone binding site.” Others also showed that for many ligands, the multiple binding sites on the HSA domains are allosterically coupled ([URL='https://www.excelmale.com/forum/javascript%3A;']72[/URL]). [I][B][COLOR=rgb(44, 130, 201)][U]It is conceivable that testosterone, like other ligands, may also have multiple binding sites with distinct affinities on HSA. Oversimplification of binding models and potentially erroneous assumptions can have major implications not only on estimates of testosterone’s bioavailability but also on putative competitive interactions with fatty acids and other hormones and drugs[/U].[/COLOR][/B] [COLOR=rgb(44, 130, 201)]The dynamics of testosterone binding to HSA requires careful reexamination using modern experimental tools. [U][B]Previous methods of comparing the solubility of testosterone in an aqueous buffer solution with its solubility in similarly buffered bovine serum albumin and using Scatchard analysis for equilibrium dialysis of testosterone with HSA were incapable of confirming multiple binding sites or identifying allosteric interactions between binding sites[/B][/U][B]. For instance, novel conformational probes that exhibit perturbations in their ground or excited-state optical properties in response to changes in their electronic environment can facilitate the characterization of the binding of hormones and drugs to HSA and evaluation of the competitive displacement by ligands. In addition, magnetic resonance spectroscopy using 13C-enriched probes can help map the spatial pockets of testosterone binding to HSA. [/B][/COLOR][/I] [B]Methods for Determination of Free Testosterone[/B] [I][COLOR=rgb(184, 49, 47)]Considering the high affinity of SHBG for testosterone binding, the SHBG-bound fraction is generally considered unavailable for biological action, and only the free and bioavailable testosterone fractions have been viewed as biologically active. [/COLOR][/I]The need for accurate assessment of free testosterone levels in the diagnosis and treatment of hypogonadism has stimulated the development of a variety of methods ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 3[/URL]), which are discussed in detail in the following sections. [B]Table 3. [COLOR=rgb(184, 49, 47)]The Relative Merits and Demerits of Various Methods of Measuring Free and Bioavailable Testosterone Levels[/COLOR][/B] [ATTACH type="full" alt="Screenshot (2360).png"]11400[/ATTACH] [ATTACH type="full" alt="Screenshot (2361).png"]11401[/ATTACH] [TABLE][TR][TH]Bioavailable Testosterone[/TH][/TR] [TR][TH][B]Method[/B][/TH] [TH][B]Merits[/B][/TH] [TH][B]Problems[/B][/TH][/TR] [TR][TD] Ammonium sulfate precipitation of SHBG-bound testosterone [/TD] [TD] • Correlates well with free testosterone obtained by equilibrium dialysis [/TD] [TD] • Technically difficult • Not easily automated • Few clinical laboratories measure it routinely •[B] [COLOR=rgb(184, 49, 47)][U]Conceptually measures non−SHBG-bound testosterone, [/U][/COLOR][U][COLOR=rgb(44, 130, 201)]which approximates but does not equal HSA-bound [/COLOR][/U][COLOR=rgb(184, 49, 47)][U]plus unbound testosterone [/U][/COLOR][/B] [/TD][/TR] [TR][TD] Concanavalin A method [/TD] [TD] • More selective and less variable than ammonium sulfate precipitation to precipitate SHBG [/TD] [TD] • Technically difficult • Not easily automated • Not used currently by clinical laboratories •[U] [COLOR=rgb(184, 49, 47)][B]Measures non−SHBG-bound testosterone, [/B][/COLOR][COLOR=rgb(44, 130, 201)][B]which approximates but does not equal HSA-bound [/B][/COLOR][/U][COLOR=rgb(184, 49, 47)][U][B]plus unbound testosterone[/B] [/U][/COLOR] [/TD][/TR] [TR][TD] [B][COLOR=rgb(184, 49, 47)]Calculated bioavailable testosterone[/COLOR][/B] [/TD] [TD] [COLOR=rgb(184, 49, 47)][B]• Based on law-of-mass-action theory or empirical equations • Simple to obtain [/B][/COLOR] [/TD] [TD] • Correlation between different algorithms is poor unless revalidated in a local laboratory • [B][COLOR=rgb(184, 49, 47)][U]Dependent on correct estimation of the association constants for the binding of testosterone to SHBG ([I]K[/I]T) and [/U][/COLOR][U][COLOR=rgb(44, 130, 201)]HSA ([I]K[/I]HSA)[/COLOR][COLOR=rgb(184, 49, 47)] • Results affected by the quality of total testosterone and SHBG and [/COLOR][COLOR=rgb(44, 130, 201)]HSA [/COLOR][/U][COLOR=rgb(184, 49, 47)][U]measurements [/U][/COLOR][/B] [/TD][/TR][/TABLE] [TABLE][TR][TH]Free Testosterone[/TH][/TR] [TR][TD] [B][COLOR=rgb(184, 49, 47)]Equilibrium dialysis [/COLOR][/B] [/TD] [TD] [B][COLOR=rgb(184, 49, 47)]• The reference method against which other methods are compared[/COLOR][/B] [/TD] [TD] [COLOR=rgb(184, 49, 47)][B]• [U]Technically difficult; operations in which the dialysis is performed vary across laboratories, contributing to high interlaboratory variability • Not easily automated • Few hospitals clinical laboratories perform this assay • Expensive • Relies on accuracy and precision of total testosterone[/U][/B][/COLOR] [/TD][/TR] [TR][TD] Ultracentrifugation [/TD] [TD] • Comparable to equilibrium dialysis [/TD] [TD] • Technically difficult • Not easily automated • Few clinical laboratories measure it routinely • Expensive • Relies on accuracy and precision of total testosterone [/TD][/TR] [TR][TD] Free androgen index [/TD] [TD] • Represents the ratio of total testosterone/SHBG • Has been shown to correlate with free testosterone measurements • Simple to obtain [/TD] [TD] • Overly simplistic and inaccurate measure of free testosterone concentrations • Poor indicator of gonadal status • Dependent on accurate measurements of total testosterone and SHBG • Most experts do not favor its use [/TD][/TR] [TR][TD] Analogue immunoassays [/TD] [TD] • Commercially available kits • High throughput and precision • Has been shown to correlate with free testosterone measurements [/TD] [TD] • Provides inaccurate estimates of free testosterone • Experts recommend against the use of direct analog assays for the measurement of free testosterone. [/TD][/TR] [TR][TD] Salivary testosterone [/TD] [TD] • Simple to obtain [/TD] [TD] • May not be an accurate marker of circulating free testosterone concentrations • Affected by sample desiccation, contamination by food and blood [/TD][/TR] [TR][TD] [B][COLOR=rgb(184, 49, 47)]Calculated free testosterone [/COLOR][/B] [/TD] [TD] [B][COLOR=rgb(184, 49, 47)]• Easy to use algorithms based on various models of testosterone binding to SHBG or empirical equations • Simple to obtain [/COLOR][/B] [/TD] [TD] • [B][COLOR=rgb(184, 49, 47)][U]Dependent upon correct estimates of the association constants and stoichiometry for binding of testosterone to SHBG and [/U][/COLOR][COLOR=rgb(44, 130, 201)][U]HSA[/U][/COLOR][COLOR=rgb(184, 49, 47)] • Accuracy and precision affected by the accuracy and precision of the total testosterone and SHBG assays [/COLOR][/B] [/TD][/TR][/TABLE] [B]Equilibrium dialysis and its various embodiments[/B] [I][COLOR=rgb(184, 49, 47)][B]Equilibrium dialysis is widely considered the reference method against which other methods are compared. It is technically demanding, and its performance is affected by assay conditions, which can result in high assay variability [/B][/COLOR][/I]([URL='https://www.excelmale.com/forum/javascript%3A;']192[/URL]). Typically, the equilibrium dialysis procedure involves the dialysis of serum or plasma samples across a semipermeable cellulose membrane with a low-molecular-weight cutoff; protein-bound testosterone is retained, whereas free testosterone equilibrates across the dialysis membrane and can be measured in the dialysate either directly using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay or immunoassay or indirectly using a tracer. Indirect methods require adding a trace amount of radioactively labeled testosterone to the sample, and after equilibrium has been achieved, the proportion of tracer in the dialysate provides a measure of the percentage of free testosterone. Because free testosterone concentration can then be calculated by multiplying the percentage of the free fraction with the total testosterone concentration obtained from the same sample in a separate assay, accurate determination of total testosterone levels is necessary for accurate determination of free testosterone levels by this method. [I][COLOR=rgb(184, 49, 47)][B][U]Although a diligently conducted equilibrium dialysis assay accurately measures free testosterone levels, the method is fraught with operator-dependent errors. The protocol itself is labor-intensive, requiring repeated purification of the radioactive tracer, and is not readily amenable to high throughput[/U].[/B][/COLOR][/I] Even some large commercial diagnostic laboratories have stopped offering this assay.[COLOR=rgb(184, 49, 47)][I] [U][B]Although equilibrium dialysis is widely considered to be the gold standard for measuring free testosterone, this method is subject to various sources of error that may contribute to inaccuracy and imprecision[/B][/U][B]. For instance, the dilution of serum or plasma may disturb the equilibrium between SHBG and its ligands ([URL='https://www.excelmale.com/forum/javascript%3A;']193[/URL]). Results may also be altered when solutes become attached to the dialysis apparatus or membrane or when there is an unequal distribution of free ligands between the two compartments as a result of[/B][/I][/COLOR][B] [COLOR=rgb(0, 0, 0)][I](1)[/I][/COLOR][COLOR=rgb(44, 130, 201)][I] inadequate time to reach equilibrium;[/I][/COLOR][COLOR=rgb(0, 0, 0)][I] (2)[/I][/COLOR][COLOR=rgb(44, 130, 201)][I] release of materials from the plate or membrane that interferes with the determination of concentration; and [/I][/COLOR][COLOR=rgb(0, 0, 0)][I](3)[/I][/COLOR][/B][COLOR=rgb(44, 130, 201)][I][B] the Donnan effect at low ionic strengths, which alters the distribution of charged particles near a semipermeable membrane so that they may not distribute evenly across the two sides of the membrane ([URL='https://www.excelmale.com/forum/javascript%3A;']194[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']195[/URL]). The ionic strength and pH of the dialysis buffer and the temperature at which dialysis is performed affect the equilibrium and the estimates of binding parameters. The batch-to-batch variability in adsorption characteristics of dialysis plates from different manufacturers may be an additional source of interassay variation. [/B][/I][/COLOR]The Centers for Disease Control and Prevention’s (CDC’s) hormone standardization program is invested in improving clinical assays and minimizing factors that affect measurement variability ([URL='https://www.excelmale.com/forum/javascript%3A;']196[/URL]). [B]Effects of temperature variations[/B] [I][COLOR=rgb(184, 49, 47)]Steroid binding is affected by the temperature and may be 2.5 times higher at 4°C than at 37°C [/COLOR][/I]([URL='https://www.excelmale.com/forum/javascript%3A;']9[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']197[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']198[/URL]). [I][COLOR=rgb(184, 49, 47)][U]The seminal testosterone-binding experiments were performed at varying temperatures—some studies were performed with ice-cold ammonium sulfate (4°C) ([URL='https://www.excelmale.com/forum/javascript%3A;']2[/URL]) or at 25°C ([URL='https://www.excelmale.com/forum/javascript%3A;']9[/URL]), which may affect binding equilibrium[/U][/COLOR][/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL]). For example, in a separate study characterizing temperature effects on cortisol protein binding by the equilibrium dialysis method, raising the temperature from 37°C to 41°C led to an increase of ∼80% in serum-free cortisol level ([URL='https://www.excelmale.com/forum/javascript%3A;']199[/URL]). [B]Effects of assay buffer composition and buffer volumes[/B] [I][COLOR=rgb(184, 49, 47)]The composition and ionic strength of the dialysis buffer affect the results of equilibrium dialysis experiments.[/COLOR][/I] [I][COLOR=rgb(184, 49, 47)][U]Experiments should ideally be performed using a dialysis buffer with an ionic composition that resembles that of human plasma, but this has not been the case in all studies. The assumption that the concentration of free ligands is equal on both sides of the membrane at equilibrium is not always valid[/U][/COLOR][/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL]). Most proteins have a charge and accumulate a set of neutralizing counterions. The Donnan effect, discussed previously, is a consequence of maintaining the overall electrical neutrality of the solution and may give spurious evidence of an association between a ligand and a protein of opposite charge when charged counterions are present in the buffer. [COLOR=rgb(184, 49, 47)][U][I]Differences in the ratios of volumes of dialysis buffer to sample may also affect estimates of free testosterone; when the binding is nonlinear, the decrease in total analyte concentration can alter the free fraction[/I][/U][/COLOR] ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL]). [B]Alteration of equilibria during physical separation of free and bound testosterone fractions[/B] [I][COLOR=rgb(184, 49, 47)]Traditional assays for determining stoichiometry and association constants usually involve separation of bound and free forms of testosterone using equilibrium dialysis, ultracentrifugation, ammonium sulfate precipitation, or other chromatographic separation methods with a subsequent Scatchard plot of the ratio of bound testosterone to unbound testosterone [(bound/free testosterone); ordinate] plotted against the bound testosterone concentration [(bound); abscissa] [/COLOR][/I]([URL='https://www.excelmale.com/forum/javascript%3A;']200[/URL]). [COLOR=rgb(184, 49, 47)][U][I]The Scatchard analysis is a method of “linearizing” data from a saturation binding experiment to determine binding constants and estimates of the stoichiometry of the noninteracting sites. However, under several experimental conditions, the underlying assumptions in the Scatchard analysis are not met, and the use of the Scatchard analysis may yield inaccurate parameter estimation[/I][/U][/COLOR] ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL]). [B][I][COLOR=rgb(184, 49, 47)][U]Achieving standardization of dialysis conditions across laboratories has been difficult, resulting in substantial interlaboratory variations in reported results. Authors who measure free testosterone by equilibrium dialysis should provide details about their methodology to ensure reproducibility and interlaboratory comparability[/U].[/COLOR][/I] Computational Algorithms for Estimating Free and Bioavailable Testosterone Concentrations: Pitfalls and the Compelling Need for Accuracy in Calculated Free Testosterone[/B] [I][COLOR=rgb(184, 49, 47)]Most hospital and commercial laboratories do not offer an equilibrium dialysis assay for free testosterone, most likely because of operational complexities in performing the assay and difficulties in automating the procedure; only a few academic and commercial laboratories offer this assay. Furthermore, efforts to standardize experimental conditions for the performance of equilibrium dialysis across the few commercial and academic laboratories that offer it have proven challenging.[/COLOR][/I] Fortunately, LC-MS/MS methods for precise total testosterone measurements and high-sensitivity SHBG enzyme-linked immunosorbent assay are widely available. [COLOR=rgb(184, 49, 47)][B][I][U]Accordingly, an accurate algorithm, validated against the equilibrium dialysis measurement, can provide calculated free testosterone values with significantly higher precision and lower cost than can be achieved with equilibrium dialysis in many hospital laboratories[/U].[/I][/B][/COLOR] Recognizing the practical difficulties that practicing clinicians face in obtaining precise and accurate measurements of free testosterone concentrations by the equilibrium dialysis method, an expert panel of the Endocrine Society concluded that “[I]calculated free testosterone, using high-quality testosterone and SHBG assays with well-defined reference intervals, is the most useful clinical marker[/I]…” ([URL='https://www.excelmale.com/forum/javascript%3A;']205[/URL]). [I][B][COLOR=rgb(184, 49, 47)]Accordingly, several groups have developed frameworks for computing free testosterone from SHBG, total testosterone, and HSA concentrations that can be broadly classified into three categories: [/COLOR][COLOR=rgb(0, 0, 0)](1) [/COLOR][COLOR=rgb(184, 49, 47)]algorithms based on linear models of testosterone binding to SHBG, [/COLOR][COLOR=rgb(0, 0, 0)](2)[/COLOR][COLOR=rgb(184, 49, 47)] algorithms derived from empiric bootstrapping of data fits to mathematical forms, and [/COLOR][COLOR=rgb(0, 0, 0)](3) [/COLOR][COLOR=rgb(184, 49, 47)][U]algorithms based on nonlinear models incorporating allostery in the SHBG dimer[/U].[/COLOR][/B][/I] [B]Calculated free testosterone based on linear models[/B] [I][COLOR=rgb(184, 49, 47)][U][B]The algorithms published by Vermeulen et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL]), Södergard et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL]), and Mazer ([URL='https://www.excelmale.com/forum/javascript%3A;']5[/URL]) are all based on the linear model of testosterone binding to SHBG [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 3(a[/URL])]. They all used Scatchard analysis to linearize data from a saturation-binding experiment to determine binding constants and estimates of the stoichiometry of noninteracting sites. However, under several experimental conditions, the underlying assumptions in the Scatchard analysis are not met, and the Scatchard analysis may yield inaccurate parameter estimation[/B][/U][B] ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 1[/URL])[/B]. For instance, the assumptions of linear regression, that the scatter of points about a line follows a normal (or Gaussian) distribution and the standard deviation is the same at every concentration of the analyte, are violated in a Scatchard plot, which alters the relationship between bound and free fractions. The use of the calculated values of the bound/free steroids further violates the assumption of linear regression that all uncertainty is in the Y variable, whereas the X variable is known with complete certainty.[/COLOR][/I] [I][COLOR=rgb(184, 49, 47)][U][B]Because of the nonlinear nature of binding and allosteric interactions between binding sites, the linear transformation of the binding data to force a straight line through nonlinear data renders these historical estimates of binding affinity and capacity prone to error[/B][/U]. Nonlinear computational tools may be more suitable for binding events, which involve allosteric interactions and are nonlinear; however, these methods have not been used in the literature for the analyses of data related to testosterone binding to SHBG or HSA.[/COLOR] [B][COLOR=rgb(184, 49, 47)][U]These algorithms use different association constants of testosterone binding to [/U][/COLOR][U][COLOR=rgb(44, 130, 201)]HSA[/COLOR][/U][COLOR=rgb(184, 49, 47)][U] and SHBG and therefore yield slightly different estimates of free testosterone[/U]. For example, the Vermeulen et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL]) equation used association constants of [/COLOR][COLOR=rgb(44, 130, 201)]3.6 × 104 [/COLOR][COLOR=rgb(184, 49, 47)]and 1 × 109 L/mol for [/COLOR][COLOR=rgb(44, 130, 201)]HSA[/COLOR][COLOR=rgb(184, 49, 47)] and SHBG, respectively, whereas the Södergard et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL]) algorithm used [/COLOR][COLOR=rgb(44, 130, 201)]4.06 × 104[/COLOR][COLOR=rgb(184, 49, 47)] and 5.97 × 108 L/ mol, respectively. All three equations, especially the Vermeulen et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL]) equation, have been widely used in the literature and in commercial laboratories. [U]The calculated free testosterone values derived using these equations correlated with free testosterone concentrations measured by equilibrium dialysis in some studies ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']214[/URL]) but displayed substantial systematic differences from values derived using the equilibrium dialysis method and from each othe[/U][/COLOR][/B][/I][COLOR=rgb(184, 49, 47)][U]r [/U][/COLOR]([URL='https://www.excelmale.com/forum/javascript%3A;']6[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']34[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']215[/URL]–[URL='https://www.excelmale.com/forum/javascript%3A;']218[/URL]). Hackbarth [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']218[/URL]) evaluated five separate equations in two patient groups with different sex distributions. They defined percentage differences above 20% to be unacceptable; depending on the equation, 32% to 72% of males and 29% to 57% of females displayed an unacceptable agreement between levels of calculated free testosterone and measured free testosterone by equilibrium dialysis. In addition, 14.9% of males and 11.1% of females showed poor fit by all five equations. [B]Calculated free testosterone from empiric and bootstrap fitting approaches[/B] [I][COLOR=rgb(184, 49, 47)]Ly and Handelsman ([URL='https://www.excelmale.com/forum/javascript%3A;']6[/URL]) analyzed a data set comprising >4000 blood samples in which free and total testosterone and SHBG concentrations were measured; dividing the data set into samples with serum total testosterone above and below 5 nM, they used a bootstrap regression modeling approach free of assumptions about theoretical binding equilibria to develop an empirical equation for free testosterone in terms of total testosterone and SHBG. Later, Sartorius et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']217[/URL]) created a variety of formulas for evaluation by bootstrap resampling to identify the best-fit model according to entropy reduction and improve upon the previous empirical calculated free testosterone equation.[/COLOR][/I] [B][I][COLOR=rgb(184, 49, 47)][U]This algorithm, like the others, is highly dependent on the accuracy and precision of the total testosterone and SHBG assays, which affects the accuracy and precision of calculated free testosterone ([URL='https://www.excelmale.com/forum/javascript%3A;']219[/URL]). Furthermore, regression equations derived empirically in one patient population may not necessarily apply to another population, especially to a population with substantially different SHBG concentrations. In a different patient population, there is no reason to believe that best-fit parameters will be the same as in the test population. [/U][/COLOR][COLOR=rgb(44, 130, 201)][U]In addition, these methods do not have a testable binding model that can be subjected to experimental validation or improved upon to incorporate other variables or new knowledge of the dynamics of testosterone binding to its cognate binding proteins or for personalization to specific conditions or disease states[/U].[/COLOR][/I] Calculated free testosterone using an algorithm that incorporates experimentally observed nonlinear binding dynamics and allosteric interaction between binding sites[/B] We recently investigated the source of systematic discrepancies between free testosterone values computed using the simple linear model, which formed the basis of the Vermeulen [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL]), Södergard [I]et al.[/I] ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL]), and Mazer ([URL='https://www.excelmale.com/forum/javascript%3A;']5[/URL]) equations, and free testosterone measured using equilibrium dialysis. [I][COLOR=rgb(184, 49, 47)][B][U]These discrepancies between free testosterone calculated using these linear binding models and free testosterone measured using equilibrium dialysis are most likely the result of the erroneous assumptions of the dynamics of testosterone binding to SHBG[/U]. Recent studies of testosterone binding to SHBG using modern biophysical techniques suggest that SHBG circulates as a homodimer and that there is complex allosteric interaction between the two binding sites on the SHBG dimer, [U]such that the binding affinities of the two sites are not identical ([URL='https://www.excelmale.com/forum/javascript%3A;']34[/URL]). The computational algorithm based on this novel multistep ensemble allosteric model (EAM)[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']34[/URL]) of testosterone binding to SHBG provided estimates of free testosterone levels that closely matched free testosterone levels measured using the equilibrium dialysis method in samples derived from men and women in two randomized clinical trials ([URL='https://www.excelmale.com/forum/javascript%3A;']220[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']221[/URL]).[/B][/COLOR][/I] [I][B][COLOR=rgb(184, 49, 47)][U]The calculated free testosterone level obtained using the prevailing linear model was systematically lower than those measured by equilibrium dialysis. In [URL='https://www.excelmale.com/forum/javascript%3A;']Table 4[/URL], we show that calculated free testosterone values across age deciles in the Framingham Heart Study computed by the linear model are lower than those computed by the allosteric model.[/U][/COLOR][COLOR=rgb(44, 130, 201)][U] The EAM model is based on experimentally derived binding affinity and dynamics, which can be verified experimentally and improved upon with additional information about other variables that determine free testosterone concentrations[/U].[/COLOR][/B][/I] [B]Lack of Standardization of Free Testosterone Measurement Methods and Unavailability of Harmonized Reference Ranges for Free Testosterone[/B] [I][COLOR=rgb(184, 49, 47)]Le et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']222[/URL]) surveyed 120 academic and community laboratories in the United States to characterize the distribution of assays and the associated reference values for free testosterone. In all, 84% of the surveyed laboratories sent their samples for free testosterone measurement to larger centralized reference laboratories ([URL='https://www.excelmale.com/forum/javascript%3A;']222[/URL]). These large commercial laboratories offered a variety of methods, including ultracentrifugation, radioimmunoassay, and calculation-based algorithms, as well as equilibrium dialysis ([URL='https://www.excelmale.com/forum/javascript%3A;']222[/URL]). Many clinical laboratories used calculated free testosterone based on published linear equations ([URL='https://www.excelmale.com/forum/javascript%3A;']3[/URL]).[/COLOR][B] [COLOR=rgb(184, 49, 47)][U]The laboratories reported wide variations in the reference ranges. [/U][/COLOR][COLOR=rgb(44, 130, 201)][U]Only 30 of the laboratories surveyed would confirm that validation studies had been performed,[/U][/COLOR][COLOR=rgb(184, 49, 47)][U] and the authors advised that reference ranges provided by manufacturers and laboratories should be interpreted with caution[/U]. [U]In a survey of 12 academic laboratories, 12 community medical laboratories, and one national laboratory, Lazarou et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']223[/URL]) found 17 and 13 different sets of reference values for total and free testosterone, respectively, which were established largely without clinical considerations[/U].[/COLOR] [COLOR=rgb(44, 130, 201)]Recently, Bhasin et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']224[/URL]) reported reference ranges for calculated free testosterone concentrations in a large, rigorously collected sample of community-dwelling men. In healthy young men of the Framingham Heart Study who were 19 to 40 years of age, the lower limit of the normal range, defined as the 2.5th percentile of calculated free testosterone, was 70 pg/mL (242.7 pmol/L) ([URL='https://www.excelmale.com/forum/javascript%3A;']198[/URL]).[/COLOR][/B][/I] [B]Clinical Implications and Recommendations[/B] Male hypogonadism is a clinical condition characterized by the presence of typical signs and symptoms in the setting of consistently low serum testosterone concentrations.[I][B] [COLOR=rgb(184, 49, 47)][U]The Endocrine Society guidelines currently suggest measuring free testosterone levels in men in whom total testosterone concentrations are near the lower limit of the normal range and in men with conditions that affect SHBG concentrations and render total testosterone a less reliable index of gonadal function[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']206[/URL]). If the free hormone hypothesis is correct, free testosterone should serve as the benchmark for biochemical confirmation of hypogonadism. [/COLOR][COLOR=rgb(44, 130, 201)][U]Accurate determination of free testosterone values is therefore central to an accurate diagnosis of hypogonadism[/U][/COLOR][COLOR=rgb(184, 49, 47)].[/COLOR][/B][/I] [COLOR=rgb(26, 188, 156)][B][I][U]The direct analog assays for free testosterone determination are inaccurate and should not be used.[/U] [/I][/B][/COLOR][I][B][COLOR=rgb(26, 188, 156)]However, a confluence of factors related to the regulatory process, economic considerations, and difficulties in performing equilibrium dialysis methods in many hospital laboratories has led to their surprising endurance despite their known inaccuracy.[/COLOR][/B][/I] Historically, laboratory-certifying bodies, such as the Clinical Laboratory Improvement Amendments, have certified laboratories and assays mostly on the basis of process measures; unlike the CDC and its Hormone Assay Standardization program for testosterone, these bodies have generally not required accuracy-based benchmarks. Similarly, the requirement in the assay approval process for demonstration of comparability to a previously approved assay enables new tracer analog assays to be approved because they can demonstrate comparability to previously approved analog methods. [B][I][COLOR=rgb(184, 49, 47)]Equilibrium dialysis is the reference method for free testosterone determination, but this assay is not always available to clinicians in all hospital laboratories; in addition, t[U]here are substantial interlaboratory variations because of the lack of standardization of assay conditions, making it difficult for practicing endocrinologists to interpret free testosterone levels[/U]. [/COLOR][/I][/B][COLOR=rgb(44, 130, 201)][I][B][U]Mechanisms to harmonize the equilibrium dialysis procedure across laboratories are needed. Until equilibrium dialysis methods can be standardized across laboratories, a computational framework that accurately captures the dynamics of testosterone to SHBG and HSA interactions in calculating free testosterone values is an unmet need for precise clinical diagnosis[/U]. The EAM appears to be an accurate and testable model for calculating free testosterone levels, but this model needs further validation in large populations.[/B][/I][/COLOR] [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
Allosterically coupled multi-site binding of T to human serum albumin
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