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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
Reference Intervals for Free Testosterone in Adult Men Measured Using a Standardized Equilibrium Dialysis Procedure
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<blockquote data-quote="madman" data-source="post: 236891" data-attributes="member: 13851"><p>Everyone jumping to conclusions already yet as I have stated numerous times there is much more to come.</p><p></p><p>Going to be a while before the s**t show comes to an end!</p><p></p><p>Fiers 2018/Jasuja 2022 paper.</p><p></p><p>Although both direct ED methods for testing FT were performed under standardized conditions the ED devices used were different.</p><p></p><p>Would not get too caught up on just the temperature/incubation times as there is much more going on.</p><p></p><p>Remember buffer composition, incubation time, and temperature will have a big impact on the free testosterone concentrations.</p><p></p><p>Throw in other assay conditions too!</p><p></p><p>Not sure what buffer composition was used in Fiers study.</p><p></p><p>As you can see the membrane MWCO (KDa) was different let alone the type of system.</p><p></p><p></p><p>ED Device <strong> Fiers 2018</strong></p><p>Description <strong>Harvard Apparatus <u>Micro-Equillibrium</u> Dialyzer System, 1 mL cells</strong></p><p>Maximum Serum Capacity (mL) <strong> 1 mL</strong></p><p>Ratio of sample/buffer volume <strong> ?</strong></p><p>Membrane Composition <strong>RC</strong></p><p>Membrane MWCO (KDa) <strong>25</strong></p><p>Incubation Time (hr) <strong>24 hrs</strong></p><p>Type of System <strong>Individual unit, assembly required</strong></p><p>Automation compatible <strong>No</strong></p><p></p><p></p><p></p><p>ED Device <strong>Jasuja 2022</strong></p><p>Description <strong>Harvard Apparatus <u>DispoEquillibrium</u> Dialyzers</strong></p><p><strong>Maximum Serum Capacity (mL) 0.2 mL</strong></p><p>Ratio of sample/buffer volume <strong> 1:1</strong></p><p>Membrane Composition <strong>RC</strong></p><p>Membrane MWCO (KDa) <strong>10</strong></p><p>Incubation Time (hr) <strong>24 hrs</strong></p><p>Type of System <strong>96-well built-in plate</strong></p><p>Automation compatible <strong> Difficult</strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong>Jasuja 2022</strong> (<a href="https://onlinelibrary.wiley.com/doi/abs/10.1111/andr.13310" target="_blank">https://onlinelibrary.wiley.com/doi/abs/10.1111/andr.13310</a>)</p><p></p><p>*<em><strong>Equilibrium dialysis was performed in 96-well plates (Harvard Apparatus, Holliston, MA) with semi-permeable membranes that allow species less than 10 kDa to pass through (<u>41,43</u>)</strong></em></p><p></p><p></p><p><strong>41. </strong>Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200.</p><p></p><p><strong>43.</strong> Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414</p><p></p><p></p><p></p><p></p><p>This is key!</p><p></p><p><em><strong><em>*Our normative ranges are similar to those reported by pioneers of this field using the legacy method (2) but the <u>percent free testosterone in our reference sample differs from that reported by another laboratory (10,52); this difference in percent free testosterone from that reported by another research laboratory (<2.8%) could be due to differences in buffer composition or in other assay conditions that are not apparent in the published methods</u> <strong>(<u>10,52</u>).</strong> The normative ranges of most commercial laboratories have changed substantially in recent years suggesting changes in their procedures over time (53,54); because procedures used by the commercial laboratories and the details of how reference ranges were derived are not published, an evaluation of these procedures was not feasible.</em></strong></em></p><p></p><p></p><p><strong>10.</strong> Vermeulen A, Kaufman JM, Giagulli VA. Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. J Clin Endocrinol Metab. 1996;81(5):1821-1826.</p><p></p><p><strong>52.</strong> Fiers T, Wu F, Moghetti P, Vanderschueren D, Lapauw B, Kaufman JM. Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis. J Clin Endocrinol Metab. 2018;103(6):2167- 2174</p><p></p><p></p><p></p><p></p><p><strong>Fiers 2018 </strong>[URL unfurl="true"]https://academic.oup.com/jcem/article/103/6/2167/4956600)[/URL]</p><p></p><p>*<em><strong>ED was performed using Fast Micro-Equilibrium dialyzer cartridges and regenerated cellulose 25 kDa membranes (Harvard Apparatus, Holliston, MA).</strong></em></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p><strong>Jasuja 2022 paper: </strong></p><p></p><p><em><strong>In an effort to generate reference intervals for free testosterone concentrations in men that can be used across laboratories, we report here a <u>detailed description of a non-proprietary, standardized equilibrium dialysis procedure</u> that can be replicated in any qualified laboratory using commercially available reagents. </strong>Instead of using a tritium-labeled tracer, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to directly measure testosterone concentrations on both sides of the dialysis chamber. <strong>We employed <u>standardized dialysis conditions at 37ºC for a duration of time shown to be sufficient for reaching equilibrium</u>. Using this <u>validated equilibrium dialysis method,</u> we evaluated the distribution of free testosterone concentrations in a prospectively-collected sample of community-living healthy nonobese men, 19 years or older. </strong>The blood samples were collected in the morning before 10 AM after an overnight fast. Total testosterone levels in the serum as well as in the dialysate were measured using a validated LC-MS/MS assay that is certified by the Hormone Standardization Program for Testosterone (HoST) of the US Centers for Disease Control and Prevention (42). We report the distribution of free testosterone concentrations in healthy young men, 19 to 39 years, as well by age groups, generated using this standardized equilibrium dialysis procedure.</em></p><p><em></em></p><p><em><strong>Free testosterone concentrations in human serum samples were determined using a standardized protocol of equilibrium dialysis coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for testosterone.</strong> <strong><u>Equilibrium dialysis was performed in 96-well plates (Harvard Apparatus, Holliston, MA) with semi-permeable membranes that allow species less than 10 kDa to pass through (41,43)</u>. The dialysis buffer composition simulated the ionic strength conditions of the human plasma:</strong> 90 mM NaCl, 3 nM KCl, 1.3 mM KH2PO4, 1.9 mM, 1.1 mM MgSO4·7H2O, 5 mM 0.30 g urea and 23 mM HEPES sodium salt, 30 nM HEPES acid, 8 mM sodium azide, and 1 mL of 0.06% DL lactic acid. 200 µL of dialysis buffer was added to the “buffer side” and 200 µL of undiluted serum was added to the “sample side”.</em></p><p></p><p></p><p><strong>41.</strong> Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200.</p><p></p><p><strong>43.</strong> Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414.</p><p></p><p></p><p></p><p></p><p><em><strong>To standardize the dialysis procedure, in preliminary experiments, we determined the time required to achieve equilibrium in the dialysis and compared the effect of incubation at room temperature versus 37ºC. After the dialysis conditions had been standardized in these preliminary experiments, the <u>dialysis plates were incubated for 24 hours at 37ºC, after which 150 µL aliquots were removed from each side for testosterone measurement using a validated LC-MS/MS assay that is certified by the Center for Disease Control’s Hormone Standardization Program (HoST) (44,45</u>). </strong>The dialysis plates were rotated throughout the course of the experiment to disrupt the formation of the Nernst diffusion layer and minimize its influence on the diffusion rate.</em></p><p></p><p></p><p><strong>44. </strong>Snyder PJ, Bhasin S, Cunningham GR, Matsumoto AM, Stephens-Shields AJ, Cauley JA, Gill TM, Barrett-Connor E, Swerdloff RS, Wang C, Ensrud KE, Lewis CE, Farrar JT, Cella D, Rosen RC, Pahor M, Crandall JP, Molitch ME, Cifelli D, Dougar D, Fluharty L, Resnick SM, Storer TW, Anton S, Basaria S, Diem SJ, Hou X, Mohler ER, 3rd, Parsons JK, Wenger NK, Zeldow B, Landis JR, Ellenberg SS, Testosterone Trials I. Effects of Testosterone Treatment in Older Men. N Engl J Med. 2016;374(7):611-624.</p><p></p><p><strong>45. </strong>Travison TG, Vesper HW, Orwoll E, Wu F, Kaufman JM, Wang Y, Lapauw B, Fiers T, Matsumoto AM, Bhasin S. Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe. J Clin Endocrinol Metab. 2017;102(4):1161-1173.</p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p>Again when it comes to the confusion of the currently used/relied upon ED methods between laboratories for testing free testosterone everyone needs to keep in mind that not all ED devices/procedures are the same.</p><p></p><p>Sums up the s**tshow!</p><p></p><p></p><p><strong>Jasuja paper:</strong></p><p></p><p><em>*An expert panel of the Endocrine Society (9) reviewed the various methods for determining free testosterone (4,5,9,21-32) and concluded that each method has some inherent limitations but that the equilibrium dialysis method is the reference standard against which all other methods should be compared (33).<strong> However, <u>substantial heterogeneity in the procedures used by various laboratories for performing the equilibrium dialysis assay</u> has contributed to variability in the reported free testosterone values</strong></em></p><p><em></em></p><p><em><em><strong>*Furthermore, the determination of free testosterone concentration by equilibrium dialysis is affected greatly by the <u>assay conditions, including the buffer composition, the incubation time, and the temperature</u></strong></em></em></p><p><em><em></em></em></p><p><em><em>*<em>The accuracy and precision of the total testosterone assay also affect the precision and accuracy of the measured free testosterone concentration. <strong><u>Most commercial laboratories do not report the buffer composition and other dialysis conditions which renders it difficult to evaluate their methods; the procedures for equilibrium dialysis have varied even in published reports from academic research laboratories (2,24,32-37)</u>. Because of the wide variation in the equilibrium dialysis procedures, the reference ranges are not generalizable across laboratories</strong></em></em></em></p><p></p><p></p><p><strong>22.</strong> Morley JE, Patrick P, Perry HM, 3rd. Evaluation of assays available to measure free testosterone. Metabolism: clinical and experimental. 2002;51(5):554-559.</p><p></p><p><strong>24.</strong> Sinha-Hikim I, Arver S, Beall G, Shen R, Guerrero M, Sattler F, Shikuma C, Nelson JC, Landgren BM, Mazer NA, Bhasin S. The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women. J Clin Endocrinol Metab. 1998;83(4):1312-1318.</p><p></p><p><strong>32.</strong> Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem. 1982;16(6):801-810.</p><p></p><p><strong>33.</strong> Ly LP, Handelsman DJ. Empirical estimation of free testosterone from testosterone and sex hormone-binding globulin immunoassays. Eur J Endocrinol. 2005;152(3):471-478.</p><p></p><p><strong>34. </strong>Hammond GL, Nisker JA, Jones LA, Siiteri PK. Estimation of the percentage of free steroid in undiluted serum by centrifugal ultrafiltration-dialysis. J Biol Chem. 1980;255(11):5023-5026.</p><p></p><p><strong>35.</strong> Pirke KM, Doerr P. Age related changes in free plasma testosterone, dihydrotestosterone and oestradiol. Acta Endocrinol (Copenh). 1975;80(1):171-178.</p><p></p><p><strong>36.</strong> Umstot ES, Baxter JE, Andersen RN. A theoretically sound and practicable equilibrium dialysis method for measuring percentage of free testosterone. J Steroid Biochem. 1985;22(5):639-648.</p><p></p><p><strong>37.</strong> Swerdloff RS, Wang C, Cunningham G, Dobs A, Iranmanesh A, Matsumoto AM, Snyder PJ, Weber T, Longstreth J, Berman N. Long-term pharmacokinetics of transdermal testosterone gel in hypogonadal men. J Clin Endocrinol Metab. 2000;85(12):4500-4510.</p></blockquote><p></p>
[QUOTE="madman, post: 236891, member: 13851"] Everyone jumping to conclusions already yet as I have stated numerous times there is much more to come. Going to be a while before the s**t show comes to an end! Fiers 2018/Jasuja 2022 paper. Although both direct ED methods for testing FT were performed under standardized conditions the ED devices used were different. Would not get too caught up on just the temperature/incubation times as there is much more going on. Remember buffer composition, incubation time, and temperature will have a big impact on the free testosterone concentrations. Throw in other assay conditions too! Not sure what buffer composition was used in Fiers study. As you can see the membrane MWCO (KDa) was different let alone the type of system. ED Device [B] Fiers 2018[/B] Description [B]Harvard Apparatus [U]Micro-Equillibrium[/U] Dialyzer System, 1 mL cells[/B] Maximum Serum Capacity (mL) [B] 1 mL[/B] Ratio of sample/buffer volume [B] ?[/B] Membrane Composition [B]RC[/B] Membrane MWCO (KDa) [B]25[/B] Incubation Time (hr) [B]24 hrs[/B] Type of System [B]Individual unit, assembly required[/B] Automation compatible [B]No[/B] ED Device [B]Jasuja 2022[/B] Description [B]Harvard Apparatus [U]DispoEquillibrium[/U] Dialyzers Maximum Serum Capacity (mL) 0.2 mL[/B] Ratio of sample/buffer volume [B] 1:1[/B] Membrane Composition [B]RC[/B] Membrane MWCO (KDa) [B]10[/B] Incubation Time (hr) [B]24 hrs[/B] Type of System [B]96-well built-in plate[/B] Automation compatible [B] Difficult Jasuja 2022[/B] ([URL]https://onlinelibrary.wiley.com/doi/abs/10.1111/andr.13310[/URL]) *[I][B]Equilibrium dialysis was performed in 96-well plates (Harvard Apparatus, Holliston, MA) with semi-permeable membranes that allow species less than 10 kDa to pass through ([U]41,43[/U])[/B][/I] [B]41. [/B]Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200. [B]43.[/B] Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414 This is key! [I][B][I]*Our normative ranges are similar to those reported by pioneers of this field using the legacy method (2) but the [U]percent free testosterone in our reference sample differs from that reported by another laboratory (10,52); this difference in percent free testosterone from that reported by another research laboratory (<2.8%) could be due to differences in buffer composition or in other assay conditions that are not apparent in the published methods[/U] [B]([U]10,52[/U]).[/B] The normative ranges of most commercial laboratories have changed substantially in recent years suggesting changes in their procedures over time (53,54); because procedures used by the commercial laboratories and the details of how reference ranges were derived are not published, an evaluation of these procedures was not feasible.[/I][/B][/I] [B]10.[/B] Vermeulen A, Kaufman JM, Giagulli VA. Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. J Clin Endocrinol Metab. 1996;81(5):1821-1826. [B]52.[/B] Fiers T, Wu F, Moghetti P, Vanderschueren D, Lapauw B, Kaufman JM. Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis. J Clin Endocrinol Metab. 2018;103(6):2167- 2174 [B]Fiers 2018 [/B][URL unfurl="true"]https://academic.oup.com/jcem/article/103/6/2167/4956600)[/URL] *[I][B]ED was performed using Fast Micro-Equilibrium dialyzer cartridges and regenerated cellulose 25 kDa membranes (Harvard Apparatus, Holliston, MA).[/B][/I] [B]Jasuja 2022 paper: [/B] [I][B]In an effort to generate reference intervals for free testosterone concentrations in men that can be used across laboratories, we report here a [U]detailed description of a non-proprietary, standardized equilibrium dialysis procedure[/U] that can be replicated in any qualified laboratory using commercially available reagents. [/B]Instead of using a tritium-labeled tracer, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to directly measure testosterone concentrations on both sides of the dialysis chamber. [B]We employed [U]standardized dialysis conditions at 37ºC for a duration of time shown to be sufficient for reaching equilibrium[/U]. Using this [U]validated equilibrium dialysis method,[/U] we evaluated the distribution of free testosterone concentrations in a prospectively-collected sample of community-living healthy nonobese men, 19 years or older. [/B]The blood samples were collected in the morning before 10 AM after an overnight fast. Total testosterone levels in the serum as well as in the dialysate were measured using a validated LC-MS/MS assay that is certified by the Hormone Standardization Program for Testosterone (HoST) of the US Centers for Disease Control and Prevention (42). We report the distribution of free testosterone concentrations in healthy young men, 19 to 39 years, as well by age groups, generated using this standardized equilibrium dialysis procedure. [B]Free testosterone concentrations in human serum samples were determined using a standardized protocol of equilibrium dialysis coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for testosterone.[/B] [B][U]Equilibrium dialysis was performed in 96-well plates (Harvard Apparatus, Holliston, MA) with semi-permeable membranes that allow species less than 10 kDa to pass through (41,43)[/U]. The dialysis buffer composition simulated the ionic strength conditions of the human plasma:[/B] 90 mM NaCl, 3 nM KCl, 1.3 mM KH2PO4, 1.9 mM, 1.1 mM MgSO4·7H2O, 5 mM 0.30 g urea and 23 mM HEPES sodium salt, 30 nM HEPES acid, 8 mM sodium azide, and 1 mL of 0.06% DL lactic acid. 200 µL of dialysis buffer was added to the “buffer side” and 200 µL of undiluted serum was added to the “sample side”.[/I] [B]41.[/B] Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200. [B]43.[/B] Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414. [I][B]To standardize the dialysis procedure, in preliminary experiments, we determined the time required to achieve equilibrium in the dialysis and compared the effect of incubation at room temperature versus 37ºC. After the dialysis conditions had been standardized in these preliminary experiments, the [U]dialysis plates were incubated for 24 hours at 37ºC, after which 150 µL aliquots were removed from each side for testosterone measurement using a validated LC-MS/MS assay that is certified by the Center for Disease Control’s Hormone Standardization Program (HoST) (44,45[/U]). [/B]The dialysis plates were rotated throughout the course of the experiment to disrupt the formation of the Nernst diffusion layer and minimize its influence on the diffusion rate.[/I] [B]44. [/B]Snyder PJ, Bhasin S, Cunningham GR, Matsumoto AM, Stephens-Shields AJ, Cauley JA, Gill TM, Barrett-Connor E, Swerdloff RS, Wang C, Ensrud KE, Lewis CE, Farrar JT, Cella D, Rosen RC, Pahor M, Crandall JP, Molitch ME, Cifelli D, Dougar D, Fluharty L, Resnick SM, Storer TW, Anton S, Basaria S, Diem SJ, Hou X, Mohler ER, 3rd, Parsons JK, Wenger NK, Zeldow B, Landis JR, Ellenberg SS, Testosterone Trials I. Effects of Testosterone Treatment in Older Men. N Engl J Med. 2016;374(7):611-624. [B]45. [/B]Travison TG, Vesper HW, Orwoll E, Wu F, Kaufman JM, Wang Y, Lapauw B, Fiers T, Matsumoto AM, Bhasin S. Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe. J Clin Endocrinol Metab. 2017;102(4):1161-1173. Again when it comes to the confusion of the currently used/relied upon ED methods between laboratories for testing free testosterone everyone needs to keep in mind that not all ED devices/procedures are the same. Sums up the s**tshow! [B]Jasuja paper:[/B] [I]*An expert panel of the Endocrine Society (9) reviewed the various methods for determining free testosterone (4,5,9,21-32) and concluded that each method has some inherent limitations but that the equilibrium dialysis method is the reference standard against which all other methods should be compared (33).[B] However, [U]substantial heterogeneity in the procedures used by various laboratories for performing the equilibrium dialysis assay[/U] has contributed to variability in the reported free testosterone values[/B] [I][B]*Furthermore, the determination of free testosterone concentration by equilibrium dialysis is affected greatly by the [U]assay conditions, including the buffer composition, the incubation time, and the temperature[/U][/B] *[I]The accuracy and precision of the total testosterone assay also affect the precision and accuracy of the measured free testosterone concentration. [B][U]Most commercial laboratories do not report the buffer composition and other dialysis conditions which renders it difficult to evaluate their methods; the procedures for equilibrium dialysis have varied even in published reports from academic research laboratories (2,24,32-37)[/U]. Because of the wide variation in the equilibrium dialysis procedures, the reference ranges are not generalizable across laboratories[/B][/I][/I][/I] [B]22.[/B] Morley JE, Patrick P, Perry HM, 3rd. Evaluation of assays available to measure free testosterone. Metabolism: clinical and experimental. 2002;51(5):554-559. [B]24.[/B] Sinha-Hikim I, Arver S, Beall G, Shen R, Guerrero M, Sattler F, Shikuma C, Nelson JC, Landgren BM, Mazer NA, Bhasin S. The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women. J Clin Endocrinol Metab. 1998;83(4):1312-1318. [B]32.[/B] Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem. 1982;16(6):801-810. [B]33.[/B] Ly LP, Handelsman DJ. Empirical estimation of free testosterone from testosterone and sex hormone-binding globulin immunoassays. Eur J Endocrinol. 2005;152(3):471-478. [B]34. [/B]Hammond GL, Nisker JA, Jones LA, Siiteri PK. Estimation of the percentage of free steroid in undiluted serum by centrifugal ultrafiltration-dialysis. J Biol Chem. 1980;255(11):5023-5026. [B]35.[/B] Pirke KM, Doerr P. Age related changes in free plasma testosterone, dihydrotestosterone and oestradiol. Acta Endocrinol (Copenh). 1975;80(1):171-178. [B]36.[/B] Umstot ES, Baxter JE, Andersen RN. A theoretically sound and practicable equilibrium dialysis method for measuring percentage of free testosterone. J Steroid Biochem. 1985;22(5):639-648. [B]37.[/B] Swerdloff RS, Wang C, Cunningham G, Dobs A, Iranmanesh A, Matsumoto AM, Snyder PJ, Weber T, Longstreth J, Berman N. Long-term pharmacokinetics of transdermal testosterone gel in hypogonadal men. J Clin Endocrinol Metab. 2000;85(12):4500-4510. [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
Reference Intervals for Free Testosterone in Adult Men Measured Using a Standardized Equilibrium Dialysis Procedure
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