madman
Super Moderator
The thread discusses the modulation of circulating free testosterone fraction during Testosterone Replacement Therapy (TRT) and its relationship with other hormones. Here are the main points from the discussion:
****************************************
Finally some newer data from Bhasins camp.
This 10 year project should be wrapping up soon and we should be hearing much more on where development of the TruT Algorithm is heading.
Will be interesting to see how this pans out!
www.excelmale.com
www.excelmale.com
*This Phase IIB proposal aims to continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered estradiol (E2), T, and SHBG concentrations and incorporating the interaction of E2 with T for wider commercial adoption in women in whom E2 levels vary greatly across the menstrual cycle and in TGD population.
*In the proposed Phase IIB studies, we will generate the v2.0 of TruTTM algorithm by incorporating the dynamics of the E2 induced perturbation in free T levels, validate it in men, women, and TGD populations (Aim 1) and deploy HIPAA-compliant, secure integration of the algorithm into electronic medical records (EMR) workflow
*(Aim 2). Future Directions and Commercialization potential: The phase IIB program will enable the pilot commercial deployment of a HIPAA-compliant (FDA registered) platform for commercializing the TruTTM (v2.0) algorithm embedded into electronic medical record (EMR) for wider clinical adoption.
*These studies will improve clinical care and advance our fundamental understanding of dynamic regulation of T bioavailability in diverse populations including unrepresented sexual and gender minorities.
pubmed.ncbi.nlm.nih.gov
1 BACKGROUND
The primary male hormone, testosterone, is converted to 5αdihydrotestosterone by the steroid 5α reductase enzymes, and to17β estradiol by the aromatase enzyme in the peripheral tissues.1,2 All three sex hormones—testosterone, dihydrotestosterone, andestradiol—exhibit limited solubility in aqueous environments and circulate bound primarily to hydrophobic ligand binding pockets of sex hormone binding globulin (SHBG) and albumin.3–6 SHBG is a dimeric molecule with each monomer of ∼45 kDa possessing one binding site.3,4,7–9 Albumin is a ∼63 kDa monomeric protein with multiple binding sites within the monomer.10 These hormones share common binding sites on each of the two serum binding proteins.3,10–14 Recenti n vitro experimental studies from our laboratory and others have established that the binding sites in the SHBG dimer and in albumin exhibit allosteric interaction.9,10,14
In spite of the recognition that all three hormones bind to the same binding sites on SHBG and albumin,15 it is not known whether and how the circulating concentrations of testosterone’s metabolites—dihydrotestosterone and estradiol—modulate the absolute and percent free testosterone concentrations during testosterone replacement therapy (TRT), which could influence the treatment effect. The potential influence of the changes in dihydrotestosterone and estradiol concentrations on free testosterone concentrations has generally not been considered in determining the therapeutic efficacy of testosterone or in adjusting the dose of testosterone. This issue is particularly relevant in the men with hypogonadism using transdermal testosterone gels, who experience substantially higher dihydrotestosterone levels than those using injectable testosterone esters.16–18
To address this question, we performed secondary analyses of data from the Testosterone Trials (TTrials) to assess the association of changes in total testosterone, dihydrotestosterone, and estradiol levels individually and conjointly with changes in percent free testosterone over the 12 months of testosterone treatment with changes in percent free testosterone.19 In the TTrials, older men with hypogonadism were treated with transdermal testosterone gel or placebo gel for 12 months. We used random forest models to evaluate the relation of changes in the free testosterone fraction with changes in the circulating concentrations of each of testosterone, dihydrotestosterone, and estradiol in TTrials participants receiving TRT.
2 METHODS
The design and efficacy results of the TTrials have been published.19 Briefly, the TTrials were a set of seven coordinated trials that evaluated the efficacy of TRT in several domains in older men, 65 years or older, with an average of two early morning, fasting testosterone concentration<275 ng/dL and one or more of low libido, mobility difficulty,or low vitality (summarized in Table 1). The eligible participants were allocated using minimization to receive either 1.0% transdermal testosterone gel or placebo gel daily for 1 year. The dose of testosterone was titrated based on the on-treatment testosterone levels to maintain serum testosterone level between 400 and 800 ng/dL. The data from all TTrials participants who were assigned to the testosterone or placebo arms and completed the study with documented baseline and 12-month testosterone levels were included.
The predictor hormones, serum total testosterone, estradiol, and dihydrotestosterone (DHT) levels were measured using liquid chromatography tandem mass spectrometry (LC‒MS/MS) in the Brigham Research Assay Core Laboratory, Boston, MA, USA. As described previously,19,20 lower limit of quantitation (LLOQ) of the LC‒MS/MS assay for testosterone was 1 ng/dL, linear range 1‒1000 ng/dL, and inter-assay coefficient of variations (CVs) were 7.9% at 49 ng/dL, 7.7%at 241 ng/dL, 4.4% at 532 ng/dL, and 3.3% at 1016 ng/dL. For the DHT LC‒MS/MS assay, LLOQ was 1 ng/dL, linear range 1‒100 ng/dL ,and inter-assay CVs were 6.1% at 5.2 ng/dL, 6.5% at 22.0 ng/dL, and 8.6% at 44.1 ng/dL, respectively.21 For the LC‒MS/MS assay for estradiol, LLOQ was 1 pg/mL, linear range 1‒500 pg/mL, and inter-assay CVs were 6.9% at 8 pg/mL, 7.0% at 77 pg/mL, and 4.8% at 206 pg/mL respectively.22 Free testosterone was measured by a method that uses equilibrium dialysis using a dialysis membrane with a cut off of 10,000 Daltons to separate total and free testosterone, as described and then measures testosterone in the dialysate using LC‒MS/MS.23 The interassay CVs were ±14.2% at 4.5 pg/mL, 11.2% at 51 pg/mL, and 11.5% at 185 pg/mL, respectively. Serum SHBG levels were measured using a two-site immuno-chemiluminescent assay (Beckman Instruments).19 The inter-assay CVs were 8.3%, 7.9%, and 10.9%, and intra-assay CVs were 7.3%, 7.1%, and 8.7%, respectively, in the low, medium, and highpools. The LLOQ of the assays is 0.5 nmol/L.
2.1 Statistical analysis
Secondary analyses of data from participants enrolled in the TTrials were performed to examine the relation of changes in the percent free testosterone levels with the changes in total testosterone, estradiol, and dihydrotestosterone concentrations. The data from the placebo and treatment arms were analyzed at baseline and during months 3, 6, 9, and 12 of testosterone treatment.
The distribution of percent free testosterone levels at baseline and at the end of the study was evaluated by intervention arm. Changes from baseline to month 12 by group were determined for all hormone levels. The comparison of change from baseline in hormone levels between groups was performed using non-parametric Mann‒Whitney U-test. Hypothesis testing was performed using two-sided alpha level of 0.05.
The random forest regression model was employed to flexibly accommodate interactions among continuous hormone predictors and assess non-linear relationship between changes in free testosterone and other hormones in the testosterone group, as described previously.24 The total testosterone, dihydrotestosterone, and estradiol concentrations were used as input variables to estimate percent free testosterone values. Graphical and quantitative methods were used to assess goodness-of-fit between empirical free testosterone values and predicted levels derived from random forest model. To assess robustness of the findings, data were randomly split into training and test sets (80% vs. 20%) and cross-validation and random search procedures were used to select appropriate parameters for the random forest model (see further details in Appendix). The values derived from the random forest model exhibited high level of concordance with the laboratory-measured values of percent free testosterone, confirming the robustness of the analytical model. To examine the influence of changes in total testosterone, dihydrotestosterone, and estradiol concentrations during TRT on percent free testosterone levels, we stratified the sample into low or high concentration groups based on first and third quartile of two hormones, with the third hormone treated as a predictor variable.
3 RESULTS
3.1 Baseline characteristics of the study population
The baseline characteristics of the TTrials participants by treatment arm have been reported previously.19 Briefly, 394 men in the testosterone arm were on average 72 years old, and 251 (63.6%) of them had a body mass index of 30 kg/m2 or greater. Mean ± standard deviation of circulating concentrations of SHBG, total testosterone, estradiol, and dihydrotestosterone hormone levels at baseline were 30.61 ± 17 nmol/L, 231.8 ± 63.1 ng/dL, 20.3 ± 6.7 pg/mL, and 21.2 ± 11.6 ng/dL, respectively.
3.2 Testosterone treatment increases percent free testosterone
As expected and reported previously, the circulating concentrations of total testosterone, dihydrotestosterone, estradiol, and absolute free testosterone increased from baseline to 12 months in testosterone treated men but did not change significantly from baseline in the placebo-treated men.19 The distribution of the percent free testosterone levels also was similar between the two groups at baseline (Figure 1A). After 12 months of intervention, the absolute free testosterone concentrations increased significantly in the testosterone group but not in the placebo group (Figure 1C). In addition to the increase in the absolute free testosterone concentrations, the percent free testosterone levels also significantly increased in the treatment group but not in the placebo group (Figure 1B). Testosterone treatment was also associated with significant increases in estradiol and dihydrotestosterone concentrations (Figure 1F,G). These increases in the total testosterone, dihydrotestosterone, estradiol, and absolute and percent free testosterone levels in testosterone-treated men were not accompanied by a significant change in SHBG levels as reported earlier19 (mean change from baseline: −1.39, 95% confidence interval−4.18, 1.58).
3.3 The changes in percent free testosterone are non-linearly associated with changes from baseline in testosterone, dihydrotestosterone, and estradiol concentrations
We further examined the relationship of the changes in each of testosterone, dihydrotestosterone, and estradiol concentrations with the changes in percent free testosterone (Figure 2). Note that the change depicted is the mean change, relative to baseline, of the measurements obtained at 3, 6, 9, and 12 months. As reported previously, serum total and free testosterone, dihydrotestosterone, and estradiol remained essentially unchanged from 3 to 12 months. Percent free testosterone levels increased non-linearly with the increases in each of testosterone, dihydrotestosterone, and estradiol concentrations as depicted in Figure 2A‒C, respectively. Even much smaller increases in circulating estradiol and dihydrotestosterone concentrations on a molar basis—substantially smaller than those in total testosterone—were associated with increases in percent free testosterone. For example, the increases in estradiols molar concentrations were over 100 times lower than those in total testosterone(Figure 2D).
3.4 The relation of changes in percent free testosterone with changes in sex hormone binding globulin and hormone levels
The changes from baseline in model-derived percent free testosterone during testosterone treatment were negatively associated with baseline SHBG levels (Figure 3A). Higher baseline SHBG concentrations were associated with smaller increases in percent free testosterone at average change in total testosterone (488.6 ng/dL), dihydrotestosterone (89.2 ng/dL), and estradiol (32.9 pg/mL) levels(Figure 3A).
A non-linear relationship was observed between free testosterone and on-treatment total testosterone concentrations when the concentrations of estradiol (32.9 pg/mL), SHBG (29.9 nM), and DHT (89.2 ng/dL) were fixed at the mean value. Percent free testosterone levels increased with increasing total testosterone concentrations reaching a plateau at high concentrations (Figure 3B). The percent free testosterone also was higher with higher on-treatment dihydrotestosterone and estradiol concentrations in testosterone treated men when the concentrations of the other hormones were fixed at the mean value, although these relationships were nonmonotonic (Figure 3C,D). It is notable that even though the absolute increase in estradiol molar concentrations during testosterone treatment is substantially smaller than that of testosterone, these smaller changes in molar concentrations of estradiol had substantial influence on percent free testosterone.
3.5 Relative concentrations of testosterone, DHT, and estradiol differentially affect the percent free testosterone levels
To further examine if the association of changes in percent free testosterone with changes in each of the three sex hormones was influenced by the relative changes in other two sex hormones, mean changes in percent free testosterone derived from the random forest model were plotted against changes in each sex hormone while maintaining the changes in the other two sex hormone to their 25th (low) or75th (high) percentiles (Figure 4). As shown in Figure 4A, the changes in percent free testosterone at any level of change from baseline in estradiol concentrations were greater at low dihydrotestosterone (25th percentile, 30 ng/dL) and low total testosterone (25th percentile, 261 ng/dL) than at high dihydrotestosterone (75th percentile, 126 ng/dL) and high total testosterone (75th percentile, 613 ng/dL). Similarly, the changes in percent free testosterone at any level of change from baseline in total testosterone with high change in estradiol (75th percentile, 40.5 pg/mL) and low DHT (25th percentile, 30 ng/dL) when compared with low estradiol (25th percentile, 20.2 pg/mL) and high DHT (75th percentile) (Figure 4C). The changes in percent free testosterone at various dihydrotestosterone concentrations were also influenced by the relative changes in estradiol and total testosterone levels (Figure 4B). Collectively, these data highlight the non-linear, concentration-dependent modulation of testosterone repartitioning into bound and free fractions by each of the three sex hormones.
4 DISCUSSION
We show here that testosterone treatment not only increases the absolute concentrations of total and free testosterone, but it also increases the percent free testosterone fraction. Percent free testosterone increased non-linearly with increasing testosterone concentrations when the concentrations of the other two sex hormones were kept fixed at their mean level. Similarly, increases in dihydrotestosterone and estradiol concentrations during testosterone treatment were associated with non-linear changes in percent free testosterone.The data from the present analyses suggest that the interaction of the three sex hormones with their cognate binding proteins is highly complex and dynamic and influenced by their relative circulating concentrations. Therefore, models of testosterones binding to SHBG, based on the assumption of fixed apparent binding affinity of sex hormones with SHBG, that do not consider the influence of estradiol and dihydrotestosterone on the free testosterone fraction are unlikely to provide accurate estimates of free testosterone fraction.
Even relatively small changes in absolute estradiol concentrations that were nearly two orders of magnitude lower than those in total testosterone concentrations influenced percent free testosterone. Similarly, increases in circulating DHT concentrations during testosterone treatment, which were substantially smaller than those in total testosterone were associated with non-linear increase in percent free testosterone. How can we explain the observed non-linear changes in free testosterone fraction during testosterone treatment and particularly the effect of substantially smaller changes in molar concentrations of estradiol on percent free testosterone in the presence of much larger changes in the molar concentrations of testosterone during testosterone treatment? Our previous studies of the binding of testosterone and estradiol to SHBG provided evidence of ligand-induced allosteric interactions between the two monomers, which are associated with conformational and energetic changes in each of the two monomers that could dynamically influence the apparent binding affinity and extend the range of ligand concentrations that the binding protein can bind.9,10,14 Furthermore, inter-monomeric allostery may offer a mechanism to precisely regulate the amount and release dynamics of the available free ligand in the background of a large total ligand concentration. Similar conformational repartitioning of protein populations and dynamic changes in ligand binding have been shown to be functionally important in other physiologic systems, including the tetrameric hemoglobin, which exhibits inter-subunit allostery in oxygen binding.24–26
In these analyses, we evaluated data from the TTrials in which testosterone treatment was associated with varying degrees of increase in total testosterone, dihydrotestosterone, and estradiol. Substantial variations in the relative concentrations of these sex hormones also are observed in women across puberty, during the menstrual cycle, pregnancy, menopause, and in some reproductive disorders.27 The importance of dynamic interactions between testosterone and estradiol with binding proteins also is being recognized as a modulator of the physiological outcomes during gender affirming hormone therapies in transgender people.28–31 In hyperthyroidism, thyroid hormone induced increase in SHBG levels is associated with greater reduction in free testosterone relative to free estradiol resulting in gynecomastia.32
These findings should be considered in the context of the study’s strengths and limitations. These secondary analyses utilized data from the TTrials, a large controlled clinical trial of TRT in which participants were allocated to intervention arms using minimization. The hormone levels were measured using validated LC‒MS/MS assays,19,33 of which testosterone and estradiol assays are certified by the CDC’s Hormone Standardization Program. Free testosterone levels were measured using a validated equilibrium dialysis method coupled to the measurement of free fraction using LC‒MS/MS.23 We used random forest analysis because it imposes fewer restrictions on the relationships of hormones with the outcome variable and because works well with continuous variables. However, these analyses were not pre-specified and need confirmation in additional studies of other data sets. Other ligands, such as phytoestrogens, xenosteroids, and some drugs could potentially bind to the same binding sites on SHBG and albumin and their influence was not considered in these analyses. Estradiol and dihydrotestosterone may exert other independent biologic effects that were not evaluated in these analyses.
Our finding that the estradiol, DHT, and testosterone interact to alter free testosterone fraction non-linearly suggests that in men with hypogonadism who are receiving TRT, free testosterone levels should be measured using a reliable method to guide the dose titration. The models that do not consider changes in estradiol and DHT concentrations are susceptible to error in estimating free testosterone concentrations.
5 CONCLUSIONS
Changes in testosterone, dihydrotestosterone, and estradiol concentrations alter free testosterone concentrations during testosterone treatment of older men with hypogonadism. Substantially smaller changes in estradiol and dihydrotestosterone concentrations than those observed in total testosterone during testosterone treatment influence percent free testosterone, suggesting complex dynamic interactions between these hormones and the binding proteins. These data suggest that changes in estradiol and dihydrotestosterone concentrations should be considered in evaluating response to testosterone treatment because of their differential influence on free testosterone concentrations in addition to their ability to exert other independent biologic effects. Because of these complex interactions between various sex hormones as well as other ligands with sex hormone binding globulin, direct measurements of free testosterone using a reliable assay, such as the equilibrium dialysis method, may be a superior marker of testosterone’s treatment effect.
- Free Testosterone Fraction:
- The thread emphasizes the importance of the free testosterone fraction, which is the biologically active form of testosterone.
- It explains that total testosterone levels alone may not provide a complete picture of hormonal status.
- Hormone Interactions:
- The discussion highlights the complex interplay between testosterone, dihydrotestosterone (DHT), and estradiol in regulating the free testosterone fraction.
- It suggests that these hormones can affect the binding of testosterone to Sex Hormone Binding Globulin (SHBG).
- DHT's Role:
- DHT is described as having a higher affinity for SHBG compared to testosterone.
- The thread suggests that DHT may displace testosterone from SHBG, potentially increasing the free testosterone fraction.
- Estradiol's Impact:
- The discussion mentions that estradiol can increase SHBG levels.
- Higher SHBG levels could potentially lead to a decrease in the free testosterone fraction.
- TRT Considerations:
- The thread implies that understanding these hormone interactions is crucial for optimizing TRT protocols.
- It suggests that monitoring and managing DHT and estradiol levels, alongside testosterone, may be important for achieving optimal results in TRT.
- Complexity of Hormone Balance:
- The discussion underscores the complexity of hormonal balance and the need for a comprehensive approach to hormone replacement therapy.
- It highlights that focusing solely on testosterone levels may not be sufficient for optimal treatment outcomes.
- Individual Variations:
- The thread acknowledges that individual responses to hormone modulation can vary, emphasizing the importance of personalized treatment approaches.
****************************************
Finally some newer data from Bhasins camp.
This 10 year project should be wrapping up soon and we should be hearing much more on where development of the TruT Algorithm is heading.
Will be interesting to see how this pans out!
Calculate Free Testosterone with TruT by FPT
The idea of SHBG as a sponge that sucks up large amounts of testosterone and leaves your free T low doesn't seem to be how it really works. The way I understand it, high SHBG makes your meager production (revealed by the free T) look like it's normal or even high when it isn't. The SHBG is...
www.excelmale.com
Calculate Free Testosterone with TruT by FPT
Labcorp Direct FT No, see above. The converted /adjusted FT direct is lower than dialysis tests. But still much closer to ED results than the "piss poor" TruT calculator. 10% off cFTv will typically get you quite close to dialysis. Note my SHBG has been cut roughly in half since starting...
www.excelmale.com
*This Phase IIB proposal aims to continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered estradiol (E2), T, and SHBG concentrations and incorporating the interaction of E2 with T for wider commercial adoption in women in whom E2 levels vary greatly across the menstrual cycle and in TGD population.
*In the proposed Phase IIB studies, we will generate the v2.0 of TruTTM algorithm by incorporating the dynamics of the E2 induced perturbation in free T levels, validate it in men, women, and TGD populations (Aim 1) and deploy HIPAA-compliant, secure integration of the algorithm into electronic medical records (EMR) workflow
*(Aim 2). Future Directions and Commercialization potential: The phase IIB program will enable the pilot commercial deployment of a HIPAA-compliant (FDA registered) platform for commercializing the TruTTM (v2.0) algorithm embedded into electronic medical record (EMR) for wider clinical adoption.
*These studies will improve clinical care and advance our fundamental understanding of dynamic regulation of T bioavailability in diverse populations including unrepresented sexual and gender minorities.
Modulation of circulating free testosterone fraction by testosterone, dihydrotestosterone, and estradiol during testosterone replacement therapy - PubMed
During testosterone replacement therapy of men with hypogonadism, changes in testosterone, dihydrotestosterone, and estradiol concentrations each altered percent free testosterone non-linearly. Small changes in estradiol concentrations exerted much larger effect on the free testosterone fraction...
pubmed.ncbi.nlm.nih.gov
1 BACKGROUND
The primary male hormone, testosterone, is converted to 5αdihydrotestosterone by the steroid 5α reductase enzymes, and to17β estradiol by the aromatase enzyme in the peripheral tissues.1,2 All three sex hormones—testosterone, dihydrotestosterone, andestradiol—exhibit limited solubility in aqueous environments and circulate bound primarily to hydrophobic ligand binding pockets of sex hormone binding globulin (SHBG) and albumin.3–6 SHBG is a dimeric molecule with each monomer of ∼45 kDa possessing one binding site.3,4,7–9 Albumin is a ∼63 kDa monomeric protein with multiple binding sites within the monomer.10 These hormones share common binding sites on each of the two serum binding proteins.3,10–14 Recenti n vitro experimental studies from our laboratory and others have established that the binding sites in the SHBG dimer and in albumin exhibit allosteric interaction.9,10,14
In spite of the recognition that all three hormones bind to the same binding sites on SHBG and albumin,15 it is not known whether and how the circulating concentrations of testosterone’s metabolites—dihydrotestosterone and estradiol—modulate the absolute and percent free testosterone concentrations during testosterone replacement therapy (TRT), which could influence the treatment effect. The potential influence of the changes in dihydrotestosterone and estradiol concentrations on free testosterone concentrations has generally not been considered in determining the therapeutic efficacy of testosterone or in adjusting the dose of testosterone. This issue is particularly relevant in the men with hypogonadism using transdermal testosterone gels, who experience substantially higher dihydrotestosterone levels than those using injectable testosterone esters.16–18
To address this question, we performed secondary analyses of data from the Testosterone Trials (TTrials) to assess the association of changes in total testosterone, dihydrotestosterone, and estradiol levels individually and conjointly with changes in percent free testosterone over the 12 months of testosterone treatment with changes in percent free testosterone.19 In the TTrials, older men with hypogonadism were treated with transdermal testosterone gel or placebo gel for 12 months. We used random forest models to evaluate the relation of changes in the free testosterone fraction with changes in the circulating concentrations of each of testosterone, dihydrotestosterone, and estradiol in TTrials participants receiving TRT.
2 METHODS
The design and efficacy results of the TTrials have been published.19 Briefly, the TTrials were a set of seven coordinated trials that evaluated the efficacy of TRT in several domains in older men, 65 years or older, with an average of two early morning, fasting testosterone concentration<275 ng/dL and one or more of low libido, mobility difficulty,or low vitality (summarized in Table 1). The eligible participants were allocated using minimization to receive either 1.0% transdermal testosterone gel or placebo gel daily for 1 year. The dose of testosterone was titrated based on the on-treatment testosterone levels to maintain serum testosterone level between 400 and 800 ng/dL. The data from all TTrials participants who were assigned to the testosterone or placebo arms and completed the study with documented baseline and 12-month testosterone levels were included.
The predictor hormones, serum total testosterone, estradiol, and dihydrotestosterone (DHT) levels were measured using liquid chromatography tandem mass spectrometry (LC‒MS/MS) in the Brigham Research Assay Core Laboratory, Boston, MA, USA. As described previously,19,20 lower limit of quantitation (LLOQ) of the LC‒MS/MS assay for testosterone was 1 ng/dL, linear range 1‒1000 ng/dL, and inter-assay coefficient of variations (CVs) were 7.9% at 49 ng/dL, 7.7%at 241 ng/dL, 4.4% at 532 ng/dL, and 3.3% at 1016 ng/dL. For the DHT LC‒MS/MS assay, LLOQ was 1 ng/dL, linear range 1‒100 ng/dL ,and inter-assay CVs were 6.1% at 5.2 ng/dL, 6.5% at 22.0 ng/dL, and 8.6% at 44.1 ng/dL, respectively.21 For the LC‒MS/MS assay for estradiol, LLOQ was 1 pg/mL, linear range 1‒500 pg/mL, and inter-assay CVs were 6.9% at 8 pg/mL, 7.0% at 77 pg/mL, and 4.8% at 206 pg/mL respectively.22 Free testosterone was measured by a method that uses equilibrium dialysis using a dialysis membrane with a cut off of 10,000 Daltons to separate total and free testosterone, as described and then measures testosterone in the dialysate using LC‒MS/MS.23 The interassay CVs were ±14.2% at 4.5 pg/mL, 11.2% at 51 pg/mL, and 11.5% at 185 pg/mL, respectively. Serum SHBG levels were measured using a two-site immuno-chemiluminescent assay (Beckman Instruments).19 The inter-assay CVs were 8.3%, 7.9%, and 10.9%, and intra-assay CVs were 7.3%, 7.1%, and 8.7%, respectively, in the low, medium, and highpools. The LLOQ of the assays is 0.5 nmol/L.
2.1 Statistical analysis
Secondary analyses of data from participants enrolled in the TTrials were performed to examine the relation of changes in the percent free testosterone levels with the changes in total testosterone, estradiol, and dihydrotestosterone concentrations. The data from the placebo and treatment arms were analyzed at baseline and during months 3, 6, 9, and 12 of testosterone treatment.
The distribution of percent free testosterone levels at baseline and at the end of the study was evaluated by intervention arm. Changes from baseline to month 12 by group were determined for all hormone levels. The comparison of change from baseline in hormone levels between groups was performed using non-parametric Mann‒Whitney U-test. Hypothesis testing was performed using two-sided alpha level of 0.05.
The random forest regression model was employed to flexibly accommodate interactions among continuous hormone predictors and assess non-linear relationship between changes in free testosterone and other hormones in the testosterone group, as described previously.24 The total testosterone, dihydrotestosterone, and estradiol concentrations were used as input variables to estimate percent free testosterone values. Graphical and quantitative methods were used to assess goodness-of-fit between empirical free testosterone values and predicted levels derived from random forest model. To assess robustness of the findings, data were randomly split into training and test sets (80% vs. 20%) and cross-validation and random search procedures were used to select appropriate parameters for the random forest model (see further details in Appendix). The values derived from the random forest model exhibited high level of concordance with the laboratory-measured values of percent free testosterone, confirming the robustness of the analytical model. To examine the influence of changes in total testosterone, dihydrotestosterone, and estradiol concentrations during TRT on percent free testosterone levels, we stratified the sample into low or high concentration groups based on first and third quartile of two hormones, with the third hormone treated as a predictor variable.
3 RESULTS
3.1 Baseline characteristics of the study population
The baseline characteristics of the TTrials participants by treatment arm have been reported previously.19 Briefly, 394 men in the testosterone arm were on average 72 years old, and 251 (63.6%) of them had a body mass index of 30 kg/m2 or greater. Mean ± standard deviation of circulating concentrations of SHBG, total testosterone, estradiol, and dihydrotestosterone hormone levels at baseline were 30.61 ± 17 nmol/L, 231.8 ± 63.1 ng/dL, 20.3 ± 6.7 pg/mL, and 21.2 ± 11.6 ng/dL, respectively.
3.2 Testosterone treatment increases percent free testosterone
As expected and reported previously, the circulating concentrations of total testosterone, dihydrotestosterone, estradiol, and absolute free testosterone increased from baseline to 12 months in testosterone treated men but did not change significantly from baseline in the placebo-treated men.19 The distribution of the percent free testosterone levels also was similar between the two groups at baseline (Figure 1A). After 12 months of intervention, the absolute free testosterone concentrations increased significantly in the testosterone group but not in the placebo group (Figure 1C). In addition to the increase in the absolute free testosterone concentrations, the percent free testosterone levels also significantly increased in the treatment group but not in the placebo group (Figure 1B). Testosterone treatment was also associated with significant increases in estradiol and dihydrotestosterone concentrations (Figure 1F,G). These increases in the total testosterone, dihydrotestosterone, estradiol, and absolute and percent free testosterone levels in testosterone-treated men were not accompanied by a significant change in SHBG levels as reported earlier19 (mean change from baseline: −1.39, 95% confidence interval−4.18, 1.58).
3.3 The changes in percent free testosterone are non-linearly associated with changes from baseline in testosterone, dihydrotestosterone, and estradiol concentrations
We further examined the relationship of the changes in each of testosterone, dihydrotestosterone, and estradiol concentrations with the changes in percent free testosterone (Figure 2). Note that the change depicted is the mean change, relative to baseline, of the measurements obtained at 3, 6, 9, and 12 months. As reported previously, serum total and free testosterone, dihydrotestosterone, and estradiol remained essentially unchanged from 3 to 12 months. Percent free testosterone levels increased non-linearly with the increases in each of testosterone, dihydrotestosterone, and estradiol concentrations as depicted in Figure 2A‒C, respectively. Even much smaller increases in circulating estradiol and dihydrotestosterone concentrations on a molar basis—substantially smaller than those in total testosterone—were associated with increases in percent free testosterone. For example, the increases in estradiols molar concentrations were over 100 times lower than those in total testosterone(Figure 2D).
3.4 The relation of changes in percent free testosterone with changes in sex hormone binding globulin and hormone levels
The changes from baseline in model-derived percent free testosterone during testosterone treatment were negatively associated with baseline SHBG levels (Figure 3A). Higher baseline SHBG concentrations were associated with smaller increases in percent free testosterone at average change in total testosterone (488.6 ng/dL), dihydrotestosterone (89.2 ng/dL), and estradiol (32.9 pg/mL) levels(Figure 3A).
A non-linear relationship was observed between free testosterone and on-treatment total testosterone concentrations when the concentrations of estradiol (32.9 pg/mL), SHBG (29.9 nM), and DHT (89.2 ng/dL) were fixed at the mean value. Percent free testosterone levels increased with increasing total testosterone concentrations reaching a plateau at high concentrations (Figure 3B). The percent free testosterone also was higher with higher on-treatment dihydrotestosterone and estradiol concentrations in testosterone treated men when the concentrations of the other hormones were fixed at the mean value, although these relationships were nonmonotonic (Figure 3C,D). It is notable that even though the absolute increase in estradiol molar concentrations during testosterone treatment is substantially smaller than that of testosterone, these smaller changes in molar concentrations of estradiol had substantial influence on percent free testosterone.
3.5 Relative concentrations of testosterone, DHT, and estradiol differentially affect the percent free testosterone levels
To further examine if the association of changes in percent free testosterone with changes in each of the three sex hormones was influenced by the relative changes in other two sex hormones, mean changes in percent free testosterone derived from the random forest model were plotted against changes in each sex hormone while maintaining the changes in the other two sex hormone to their 25th (low) or75th (high) percentiles (Figure 4). As shown in Figure 4A, the changes in percent free testosterone at any level of change from baseline in estradiol concentrations were greater at low dihydrotestosterone (25th percentile, 30 ng/dL) and low total testosterone (25th percentile, 261 ng/dL) than at high dihydrotestosterone (75th percentile, 126 ng/dL) and high total testosterone (75th percentile, 613 ng/dL). Similarly, the changes in percent free testosterone at any level of change from baseline in total testosterone with high change in estradiol (75th percentile, 40.5 pg/mL) and low DHT (25th percentile, 30 ng/dL) when compared with low estradiol (25th percentile, 20.2 pg/mL) and high DHT (75th percentile) (Figure 4C). The changes in percent free testosterone at various dihydrotestosterone concentrations were also influenced by the relative changes in estradiol and total testosterone levels (Figure 4B). Collectively, these data highlight the non-linear, concentration-dependent modulation of testosterone repartitioning into bound and free fractions by each of the three sex hormones.
4 DISCUSSION
We show here that testosterone treatment not only increases the absolute concentrations of total and free testosterone, but it also increases the percent free testosterone fraction. Percent free testosterone increased non-linearly with increasing testosterone concentrations when the concentrations of the other two sex hormones were kept fixed at their mean level. Similarly, increases in dihydrotestosterone and estradiol concentrations during testosterone treatment were associated with non-linear changes in percent free testosterone.The data from the present analyses suggest that the interaction of the three sex hormones with their cognate binding proteins is highly complex and dynamic and influenced by their relative circulating concentrations. Therefore, models of testosterones binding to SHBG, based on the assumption of fixed apparent binding affinity of sex hormones with SHBG, that do not consider the influence of estradiol and dihydrotestosterone on the free testosterone fraction are unlikely to provide accurate estimates of free testosterone fraction.
Even relatively small changes in absolute estradiol concentrations that were nearly two orders of magnitude lower than those in total testosterone concentrations influenced percent free testosterone. Similarly, increases in circulating DHT concentrations during testosterone treatment, which were substantially smaller than those in total testosterone were associated with non-linear increase in percent free testosterone. How can we explain the observed non-linear changes in free testosterone fraction during testosterone treatment and particularly the effect of substantially smaller changes in molar concentrations of estradiol on percent free testosterone in the presence of much larger changes in the molar concentrations of testosterone during testosterone treatment? Our previous studies of the binding of testosterone and estradiol to SHBG provided evidence of ligand-induced allosteric interactions between the two monomers, which are associated with conformational and energetic changes in each of the two monomers that could dynamically influence the apparent binding affinity and extend the range of ligand concentrations that the binding protein can bind.9,10,14 Furthermore, inter-monomeric allostery may offer a mechanism to precisely regulate the amount and release dynamics of the available free ligand in the background of a large total ligand concentration. Similar conformational repartitioning of protein populations and dynamic changes in ligand binding have been shown to be functionally important in other physiologic systems, including the tetrameric hemoglobin, which exhibits inter-subunit allostery in oxygen binding.24–26
In these analyses, we evaluated data from the TTrials in which testosterone treatment was associated with varying degrees of increase in total testosterone, dihydrotestosterone, and estradiol. Substantial variations in the relative concentrations of these sex hormones also are observed in women across puberty, during the menstrual cycle, pregnancy, menopause, and in some reproductive disorders.27 The importance of dynamic interactions between testosterone and estradiol with binding proteins also is being recognized as a modulator of the physiological outcomes during gender affirming hormone therapies in transgender people.28–31 In hyperthyroidism, thyroid hormone induced increase in SHBG levels is associated with greater reduction in free testosterone relative to free estradiol resulting in gynecomastia.32
These findings should be considered in the context of the study’s strengths and limitations. These secondary analyses utilized data from the TTrials, a large controlled clinical trial of TRT in which participants were allocated to intervention arms using minimization. The hormone levels were measured using validated LC‒MS/MS assays,19,33 of which testosterone and estradiol assays are certified by the CDC’s Hormone Standardization Program. Free testosterone levels were measured using a validated equilibrium dialysis method coupled to the measurement of free fraction using LC‒MS/MS.23 We used random forest analysis because it imposes fewer restrictions on the relationships of hormones with the outcome variable and because works well with continuous variables. However, these analyses were not pre-specified and need confirmation in additional studies of other data sets. Other ligands, such as phytoestrogens, xenosteroids, and some drugs could potentially bind to the same binding sites on SHBG and albumin and their influence was not considered in these analyses. Estradiol and dihydrotestosterone may exert other independent biologic effects that were not evaluated in these analyses.
Our finding that the estradiol, DHT, and testosterone interact to alter free testosterone fraction non-linearly suggests that in men with hypogonadism who are receiving TRT, free testosterone levels should be measured using a reliable method to guide the dose titration. The models that do not consider changes in estradiol and DHT concentrations are susceptible to error in estimating free testosterone concentrations.
5 CONCLUSIONS
Changes in testosterone, dihydrotestosterone, and estradiol concentrations alter free testosterone concentrations during testosterone treatment of older men with hypogonadism. Substantially smaller changes in estradiol and dihydrotestosterone concentrations than those observed in total testosterone during testosterone treatment influence percent free testosterone, suggesting complex dynamic interactions between these hormones and the binding proteins. These data suggest that changes in estradiol and dihydrotestosterone concentrations should be considered in evaluating response to testosterone treatment because of their differential influence on free testosterone concentrations in addition to their ability to exert other independent biologic effects. Because of these complex interactions between various sex hormones as well as other ligands with sex hormone binding globulin, direct measurements of free testosterone using a reliable assay, such as the equilibrium dialysis method, may be a superior marker of testosterone’s treatment effect.
