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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
Gman's Test Prop Labs
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<blockquote data-quote="madman" data-source="post: 153135" data-attributes="member: 13851"><p><strong><span style="color: rgb(184, 49, 47)">2. Free E2: calculate or measure? </span></strong></p><p></p><p><strong>2.1. Calculate </strong></p><p></p><p>Substantially more effort has gone into examining the vagaries of free T than free E2. Although there are distinct differences in estimating free T and free E2, the general approaches are the same and, as appropriate, we will cite papers dealing with the approach to calculating/measuring free T. <strong>To calculate free estradiol one must first have <span style="color: rgb(184, 49, 47)">an accurate measure of total estradiol</span>, SHBG, albumin, and the appropriate <span style="color: rgb(184, 49, 47)">Ka’s.</span> <span style="color: rgb(44, 130, 201)">The difficulties with, and problems of, assaying estradiol constitute a large part of this special issue and I would simply emphasize that without a proper assay for total estradiol, there can be no accurate determination of free estradiol.</span></strong> <span style="color: rgb(147, 101, 184)"><strong>SHBG can be measured, either by its ligand binding capacity, by immunoassay or, more recently by mass spectrometry [9]. Commercial methods are available for its immunoassay. At least some of those assays have been standardized against the binding capacity of a standard SHBG preparation obtained from the WHO. There are however problems with the standard; its binding capacity is lower than the previous standards, and its Ka for the SHBG steroid interaction is also lower [10]. Further, one need be sure that the standard’s binding activity is equal to its immunologic activity as the two activities must be proven to be identical and are differentially sensitive to denaturation.</strong></span> <span style="color: rgb(184, 49, 47)"><strong>Estimation of the proper</strong></span> <span style="color: rgb(184, 49, 47)"><strong>association constants for E2-SHBG</strong></span> <span style="color: rgb(44, 130, 201)"><strong>and E2-albumin are problematic (Table 1). </strong></span><strong><span style="color: rgb(184, 49, 47)">As can be seen, there is a >3-fold difference in the highest vs. the lowest Ka for estradiol-SHBG</span> and an almost 2-fold difference in the highest vs. the lowest Ka for estradiol–albumin.</strong> <span style="color: rgb(0, 0, 0)"><strong>Thus, depending on the choice of Ka, widely discrepant values would be calculated.</strong></span> <span style="color: rgb(26, 188, 156)"><strong>Finally, it is possible for a variety of other steroids to compete for binding with E2. This would raise the concentration of free estradiol, but is not ordinarily taken into account in calculated values.</strong></span> <span style="color: rgb(0, 0, 0)"><strong>For the most part, this does not appear to be a problem in non-pregnant women (in whom competing steroids do not circulate in sufficiently high concentrations to increase free estradiol) </strong></span><span style="color: rgb(184, 49, 47)"><strong>but can yield incorrect results in the presence of unsuspected competitors or in known situations, such as male plasma, where the concentration of T far exceeds that of estradiol and displaces it from SHBG binding sites. </strong></span>If competitors are present and measured, the overall binding equations can be altered and appropriate corrections can be made [11]. Despite all these caveats, calculation is the prevalent way that free E2 is estimated. For those agreeing on the various measurements, and using the same Ka’s, the results can be comparable, thus giving the false impression of ‘‘accuracy by the majority.’’ <span style="color: rgb(44, 130, 201)"><strong>Finally, it has been suggested that the binding of ligands to SHBG is allosteric and that the two binding sites on the homodimer do not have the same Ka. </strong></span><span style="color: rgb(251, 160, 38)"><strong>Occupancy of one of the sites on the homodimer is posited to alter the Ka of the other.</strong></span> <span style="color: rgb(184, 49, 47)"><strong>Modeling based on this concept is said to result in calculated values in better agreement with careful measurements of free estradiol than previous models [12]. </strong></span></p><p></p><p></p><p>[ATTACH=full]7796[/ATTACH]</p><p></p><p></p><p><strong><span style="color: rgb(0, 0, 0)">2.2. Measure </span></strong></p><p></p><p><strong><span style="color: rgb(184, 49, 47)">As stated above, free E2 in plasma is a theoretical construct that follows from the Law of Mass Action which was formulated by chemists in the late 1800’s and ‘‘explains and predicts behaviours [sic] of solutes in dynamic equilibrium.’’ </span></strong><span style="color: rgb(44, 130, 201)"><strong>Acceptance of the Law of Mass Action does not necessarily imply that all the components of an equilibrium mixture can be accurately measured or, indeed, how it is to be done. </strong></span><strong>Whatever method is used ideally should not: disturb the equilibrium that exists in vivo; change the environment that exists in vivo (ionic strength pH, etc); and be accomplished at the same temperature that exists in vivo. The two methods that best approximate these criteria are <span style="color: rgb(251, 160, 38)">equilibrium dialysis</span> and <span style="color: rgb(250, 197, 28)">centrifugal ultrafiltration </span>(<span style="color: rgb(184, 49, 47)">see Table 2</span>) </strong></p><p></p><p></p><p>[ATTACH=full]7797[/ATTACH]</p><p></p><p></p><p><strong>2.2.3. Choose a method </strong></p><p></p><p><strong><span style="color: rgb(184, 49, 47)">The choice is guided by both convenience/ease and the desire to obtain the answer most likely to coincide with the true value of free E2.</span></strong> <span style="color: rgb(44, 130, 201)"><strong>Whichever method one chooses, E2 must be accurately and reproducibly measured; this is required whether one calculates or measures. </strong></span><strong>Once having conceded the ability to measure E2, then the calculation offers the advantages of simplicity and economy. SHBG and albumin are easily measured and the calculation is automated. </strong><span style="color: rgb(26, 188, 156)"><strong>On the negative side: there is disagreement about the proper Ka’s and SHBG is not properly standardized/harmonized. </strong></span>In spite of these potential hazards, a number of communications have claimed excellent agreement between calculated and measured values for free testosterone [13,16,20] and free estradiol [13]; however, a number of publications find disagreement between measured and calculated values [21–23]. <strong><span style="color: rgb(184, 49, 47)">In addition the calculation of free E2 has been compared to measured values, and found to be satisfactory, in only a small sample of postmenopausal patients [13]; </span></strong><span style="color: rgb(44, 130, 201)"><strong>the authors caution against using the calculated value in other populations. Moreover, it should be recalled that E2 is bound to SHBG 2–3-fold less tightly to SHBG than is testosterone, rendering the assumption of a fixed value for albumin unacceptable until the appropriate calculations and experiments are at hand. </strong></span><strong><span style="color: rgb(147, 101, 184)">This is still an unsettled issue and no irreversible recommendations as to the best way to proceed can be issued at this time.</span></strong></p><p></p><p></p><p>We need to keep this in mind</p><p>[ATTACH=full]7798[/ATTACH]</p></blockquote><p></p>
[QUOTE="madman, post: 153135, member: 13851"] [B][COLOR=rgb(184, 49, 47)]2. Free E2: calculate or measure? [/COLOR][/B] [B]2.1. Calculate [/B] Substantially more effort has gone into examining the vagaries of free T than free E2. Although there are distinct differences in estimating free T and free E2, the general approaches are the same and, as appropriate, we will cite papers dealing with the approach to calculating/measuring free T. [B]To calculate free estradiol one must first have [COLOR=rgb(184, 49, 47)]an accurate measure of total estradiol[/COLOR], SHBG, albumin, and the appropriate [COLOR=rgb(184, 49, 47)]Ka’s.[/COLOR] [COLOR=rgb(44, 130, 201)]The difficulties with, and problems of, assaying estradiol constitute a large part of this special issue and I would simply emphasize that without a proper assay for total estradiol, there can be no accurate determination of free estradiol.[/COLOR][/B] [COLOR=rgb(147, 101, 184)][B]SHBG can be measured, either by its ligand binding capacity, by immunoassay or, more recently by mass spectrometry [9]. Commercial methods are available for its immunoassay. At least some of those assays have been standardized against the binding capacity of a standard SHBG preparation obtained from the WHO. There are however problems with the standard; its binding capacity is lower than the previous standards, and its Ka for the SHBG steroid interaction is also lower [10]. Further, one need be sure that the standard’s binding activity is equal to its immunologic activity as the two activities must be proven to be identical and are differentially sensitive to denaturation.[/B][/COLOR] [COLOR=rgb(184, 49, 47)][B]Estimation of the proper[/B][/COLOR] [COLOR=rgb(184, 49, 47)][B]association constants for E2-SHBG[/B][/COLOR] [COLOR=rgb(44, 130, 201)][B]and E2-albumin are problematic (Table 1). [/B][/COLOR][B][COLOR=rgb(184, 49, 47)]As can be seen, there is a >3-fold difference in the highest vs. the lowest Ka for estradiol-SHBG[/COLOR] and an almost 2-fold difference in the highest vs. the lowest Ka for estradiol–albumin.[/B] [COLOR=rgb(0, 0, 0)][B]Thus, depending on the choice of Ka, widely discrepant values would be calculated.[/B][/COLOR] [COLOR=rgb(26, 188, 156)][B]Finally, it is possible for a variety of other steroids to compete for binding with E2. This would raise the concentration of free estradiol, but is not ordinarily taken into account in calculated values.[/B][/COLOR] [COLOR=rgb(0, 0, 0)][B]For the most part, this does not appear to be a problem in non-pregnant women (in whom competing steroids do not circulate in sufficiently high concentrations to increase free estradiol) [/B][/COLOR][COLOR=rgb(184, 49, 47)][B]but can yield incorrect results in the presence of unsuspected competitors or in known situations, such as male plasma, where the concentration of T far exceeds that of estradiol and displaces it from SHBG binding sites. [/B][/COLOR]If competitors are present and measured, the overall binding equations can be altered and appropriate corrections can be made [11]. Despite all these caveats, calculation is the prevalent way that free E2 is estimated. For those agreeing on the various measurements, and using the same Ka’s, the results can be comparable, thus giving the false impression of ‘‘accuracy by the majority.’’ [COLOR=rgb(44, 130, 201)][B]Finally, it has been suggested that the binding of ligands to SHBG is allosteric and that the two binding sites on the homodimer do not have the same Ka. [/B][/COLOR][COLOR=rgb(251, 160, 38)][B]Occupancy of one of the sites on the homodimer is posited to alter the Ka of the other.[/B][/COLOR] [COLOR=rgb(184, 49, 47)][B]Modeling based on this concept is said to result in calculated values in better agreement with careful measurements of free estradiol than previous models [12]. [/B][/COLOR] [ATTACH=full]7796[/ATTACH] [B][COLOR=rgb(0, 0, 0)]2.2. Measure [/COLOR][/B] [B][COLOR=rgb(184, 49, 47)]As stated above, free E2 in plasma is a theoretical construct that follows from the Law of Mass Action which was formulated by chemists in the late 1800’s and ‘‘explains and predicts behaviours [sic] of solutes in dynamic equilibrium.’’ [/COLOR][/B][COLOR=rgb(44, 130, 201)][B]Acceptance of the Law of Mass Action does not necessarily imply that all the components of an equilibrium mixture can be accurately measured or, indeed, how it is to be done. [/B][/COLOR][B]Whatever method is used ideally should not: disturb the equilibrium that exists in vivo; change the environment that exists in vivo (ionic strength pH, etc); and be accomplished at the same temperature that exists in vivo. The two methods that best approximate these criteria are [COLOR=rgb(251, 160, 38)]equilibrium dialysis[/COLOR] and [COLOR=rgb(250, 197, 28)]centrifugal ultrafiltration [/COLOR]([COLOR=rgb(184, 49, 47)]see Table 2[/COLOR]) [/B] [ATTACH=full]7797[/ATTACH] [B]2.2.3. Choose a method [/B] [B][COLOR=rgb(184, 49, 47)]The choice is guided by both convenience/ease and the desire to obtain the answer most likely to coincide with the true value of free E2.[/COLOR][/B] [COLOR=rgb(44, 130, 201)][B]Whichever method one chooses, E2 must be accurately and reproducibly measured; this is required whether one calculates or measures. [/B][/COLOR][B]Once having conceded the ability to measure E2, then the calculation offers the advantages of simplicity and economy. SHBG and albumin are easily measured and the calculation is automated. [/B][COLOR=rgb(26, 188, 156)][B]On the negative side: there is disagreement about the proper Ka’s and SHBG is not properly standardized/harmonized. [/B][/COLOR]In spite of these potential hazards, a number of communications have claimed excellent agreement between calculated and measured values for free testosterone [13,16,20] and free estradiol [13]; however, a number of publications find disagreement between measured and calculated values [21–23]. [B][COLOR=rgb(184, 49, 47)]In addition the calculation of free E2 has been compared to measured values, and found to be satisfactory, in only a small sample of postmenopausal patients [13]; [/COLOR][/B][COLOR=rgb(44, 130, 201)][B]the authors caution against using the calculated value in other populations. Moreover, it should be recalled that E2 is bound to SHBG 2–3-fold less tightly to SHBG than is testosterone, rendering the assumption of a fixed value for albumin unacceptable until the appropriate calculations and experiments are at hand. [/B][/COLOR][B][COLOR=rgb(147, 101, 184)]This is still an unsettled issue and no irreversible recommendations as to the best way to proceed can be issued at this time.[/COLOR][/B] We need to keep this in mind [ATTACH=full]7798[/ATTACH] [/QUOTE]
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Blood Test Discussion
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