Calculate Free Testosterone with TruT by FPT
- Thread starter madman
- Start date
I think the good doctors have recognized early on that the direct free T test results frequently don't make sense.
DragonBits
Well-Known Member
I knew over 7 years ago that the typical Free T wasn't reliable, but didn't know how confusing the situation was.
So a free T calculator or a TruT might be right, but it doesn't come with a range on what is normal. I suppose like you did I could calc a range from what I would assume is normal Total T and average SHBG/ALB levels to derive my own range.
But is it correct that most estradiol comes from FreeT and not total T?
So if one converts more or less free T to estradiol, then that in turn lowers or raises free T independent of SHBG/ALB?
I don't see anything in the calculators for how much estradiol one produces.
Presumably there should be some normal ranges built up for equilibrium dialysis tests. If so then the advantage of TruT would lie in using the same ranges, though the developers of TruT may be working to establish normals. The Vermeulen method is still useful, in that it correlates well with results of equilibrium dialysis. But the absolute numbers don't match.
My understanding is that estradiol comes from free T, not total. I don't think the rate of conversion is high enough to change free T independently of SHBG/ALB. That is, as conversion happens more T is released from ALB (fast) and SHBG (slow).
In the multi-ligand model, free estradiol is a function of testosterone, total estradiol, SHBG etc. But total estradiol production is in turn mainly a function of free testosterone and aromatase.
madman
Super Moderator
Need to look further..... thinking this will be the set reference range for the TruT calculated free testosterone method:
"Based on the new data on the distribution of free testosterone levels in healthy men the target range of free testosterone has been determined to be 164 to 314 pg/ml (mean+/−1SD)"
Which would convert to 16-31 ng/dl
madman
Super Moderator
Three heavy weights in the field behind the invention of TruT.
Ravi Jasuja, Shalender Bhasin and Mikhail N Zakharov.
Hoping people understand the significance/importance of this testing method and the huge impact it will have.
Very long read but deeply interesting to say the least WO2014138026 METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS
1. (WO2014138026) METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS
IPC:
Applicants:
Inventors:
Agent:
Priority Data:
Title(EN)METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS
(FR)PROCÉDÉS ET SYSTÈMES POUR LE DIAGNOSTIC ET LE TRAITEMENT DE TROUBLES ANDROGÉNIQUES
Abstract:
SUMMARY
[0005] Described herein is the inventors' demonstration that the prevailing model of testosterone's binding to SHBG, used in the current methods of testing and diagnosis is flawed, and their further discovery of an improved model that permits methods, assays, and systems with greater accuracy and reliability in measuring testosterone levels. The multi-step dynamic binding model with complex allostery described herein is a new model for calculation of free testosterone. The multi-step dynamic binding model with complex allostery model is a modified ensemble allostery model that takes into consideration the specific SHBG-Testosterone binding interaction described herein.
[0006] In one aspect, described herein is a computer implemented method for an assay, comprising: on a device having one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs including instructions for: a) receiving data from measuring i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration in a biological sample obtained from an individual, to determine free testosterone concentration from the individual; b) attributing at least two distinct interconverting microstates of an unliganded SHBG dimer having a first monomer and a second monomer; and c) calculating the free testosterone concentration in the individual using the New Multi-Step Dynamic Binding Model with Complex Allostery encompassing readjustment of a first equilibria between the microstates upon binding of a first testosterone molecule to the first monomer and an allosteric interaction between two binding sites of the SHBG dimer.
*Refer to article to read [0007] - [0017]
[0018] In one aspect, described herein is a method for determining a need for adjustment of a dose of testosterone administered to an individual comprising a) determining the concentration of free testosterone in an individual receiving testosterone therapy at a first dose, wherein the concentration of free testosterone is determined by b) measuring i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration in a biological sample obtained from an individual, to determine free testosterone concentration from the individual; c) attributing at least two distinct interconverting microstates of an unliganded SHBG dimer having a first monomer and a second monomer by applying the New Multi-Step Dynamic Binding Model with Complex Allostery to the data of steps a) and b); d) calculating the free testosterone concentration in the individual using the New Multi-step Dynamic Binding Model with Complex Allostery encompassing readjustment of a first equilibria between the microstates upon binding of a first testosterone molecule to the first monomer and an allosteric interaction between two binding sites of the SHBG dimer; e) providing a second dose of testosterone that is higher than the first dose when the free testosterone concentration is below the lower end of the target therapeutic range (e.g.164 pg/ml); and f) providing a second dose of testosterone that is lower than the first dose when the free testosterone concentration is above the upper end of the target therapeutic range (314 pg/ml).
[0019] In some embodiments of any of the foregoing aspects, the step of attributing can be performed according to Figures 2, 3, 5, and 7. In some embodiments, of any of the foregoing aspects, the step of calculating can be performed according to Figure 7 or Example 5. In some embodiments of any of the foregoing aspects, the individual is a male over the age of 35. In some embodiments of any of the foregoing aspects, the androgen disorder is selected from the group consisting of a testosterone deficiency, an androgen deficiency, a hyperandrogenic disorder, an androgen expressing tumor, and a hypogonadism disorder. In some embodiments of any of the foregoing aspects, the androgen disorder is a hyperandrogenic disorder selected from the group consisting of an acne disorder, a hirsutism disorder, and an androgenic alopecia disorder. In some embodiments of any of the foregoing aspects, the individual has been diagnosed with a disease selected from the group consisting of: diabetes, human immunodeficiency virus (HIV), hepatitis B, hepatitis C, hypothyroidism or hyperthyroidism, androgen insensitivity, acromegaly, anorexia, muscular dystrophy, liver disease, cancer cachexia, malnutrition, nephrotic syndrome, and obesity, and other conditions in which SHBG or albumin concentrations are altered. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of classifying the individual into categories based on additional clinical symptoms. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of using the free testosterone concentration determined using the new Multistep Dynamic Binding Model with Complex Allostery to determine the dose or to individually adjust the dose of a formulation of testosterone for the treatment of a medical disease, taking into account patient's age, body weight and body mass index, medical conditions, including any co-morbid conditions, albumin and SHBG, and/or LH and FSH concentrations, and other patient-specific factors. In some embodiments of any of the foregoing aspects, instead of steps a-c, the data received is a previously calculated concentration of free testostosterone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figs. 1A-1B demonstrate that free T values calculated using the Vermeulen/ Sodergard/ Mazer model - the commonly used method for determining free T concentrations - differ from those measured using equilibrium dialysis. Fig. 1A depicts a graph of free testosterone concentrations in samples derived from a randomized testosterone trial (The TED study) were measured using the equilibrium dialysis and plotted against those calculated using the Vermeulen/ Sodergard/ Mazer equation. Calculated free T concentrations differed systematically from the measured values. Fig. 1B depicts a Bland Altman plot revealing substantial discrepancy between the calculated and measured free T concentrations.
[0021] Figs. 2A-2C demonstrate that binding of testosterone to SHBG displays complex behavior. Fig. 2A depicts a graph demonstrating that binding isotherms display complex behavior. Graded concentrations of testosterone were incubated overnight with various SHBG concentrations and the amount of bound testosterone was plotted against total T (added) concentrations (squares) 20nM, (triangles) 10 nM, (circles) 5nM SHBG. The fit curves represent the result of the linked fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery (Fig. 3). Fig. 2B depicts a graph of the depletion of free testosterone by varying SHBG concentration which is best described by the new Multi-step Dynamic Binding Model with Complex Allostery. Shown is concentration of free testosterone in the equilibrium dialysis experiment. One side of the equilibrium dialysis membrane has varying concentration of SHBG in buffer, the other one has plain buffer. Constant concentration of testosterone is added to each well of multi-well dialyzer: (triangles) 8.7nM. Curves are the result of the linked fit of data in Panel B to the new Multi-step Dynamic Binding Model with Complex Allostery (Fig 3).
Fig. 2C depicts a graph of the heat of T:SHBG association measured by isothermal calorimetry (ITC). Presented is the integrated ITC curve with buffer heats subtracted. SHBG starting concentration is 5μΜ. Experimental points are shown by squares, fit to the model in Fig.3 is shown by a solid line.
[0022] Fig. 3 depicts a schematic representation of the new Multi-step Dynamic Binding Model with Complex Allostery of testosterone's binding to SHBG and albumin, developed in this study. Unliganded SHBG dimers (S2, S2') exist in conformational equilibrium. Upon binding of the first testosterone molecule to microstates S2 and S2' can result in conformationally heterogeneous intermediate states S2T and S2'T respectively. These singly-occupied microstates then converge to S2T2 upon binding of the second testosterone molecule.
[0023] Figs. 4A-4B demonstrate a comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation (23) as implemented by Mazer (24) or the New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery with those measured using the equilibrium dialysis in samples from the 5alpha reductase trial. Fig. 4A depicts a graph of a comparison of the free testosterone concentration calculated by the Vermeulen equation and the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery to that measured by equilibrium dialysis in samples from a randomized testosterone trial in men. (■) free testosterone concentrations derived using an algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery; (•) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24). Solid lines are lines of best linear fit. Regression lines fit new Multi-step Dynamic Binding Model with Complex Allostery calculation (slope = 1.01±0.01, regression line fitting the squares), and the Vermeulen model (slope 0.77±0.02, lower line fitting the dots). Magenta dashed line is the line of prefect correlation. Fig. 4B depicts Bland Altman plots of the relative frequency distribution of % difference of calculated and measured free testosterone using either the Vermeulen equation (squares) or the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery (black dots) The relative deviations from the measured value are distributed around 0 for new Multi-step Dynamic Binding Model with Complex Allostery model and are different from zero for the Vermeulen model.
Figs. 4C-4D depict a Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation or the New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery with those measured using the equilibrium dialysis. Figs. 4C-4D demonstrate a Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation or the New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery with those measured using the equilibrium dialysis in samples from a randomized testosterone trial in men with ED. Fig. 4C depicts a graph of a comparison of the free testosterone concentration calculated by the Vermeulen equation and the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery to that measured by equilibrium dialysis in samples from a randomized testosterone trial in men. (squares) free testosterone concentrations derived using an algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery; (circles) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24). Black regression line fits new Multi-step Dynamic Binding Model with Complex Allostery model calculation (slope = 1.01±0.01 is shown) Fig. 4D depicts Bland Altman plots of the relative frequency of % difference of calculated and measured free testosterone using either the Vermeulen equation or the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery. The relative deviations from the measured value are distributed around 0 for the new Multi-step Dynamic Binding Model with Complex Allostery model and are different from zero for the Vermeulen model.
[0024] Fig. 5A depicts a graph of the binding isotherm. Graded concentrations of testosterone were incubated overnight with 20 nM SHBG and the amount of bound testosterone was plotted against total testosterone concentration. The fit curve represents the fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery. Fig. 5B depicts a graph of the depletion curve. A constant concentration of testosterone (8.7 nM) was incubated with increasing SHBG concentrations. Free testosterone concentration is plotted against SHBG concentration. Solid line represents the fit of data to new Multi-step Dynamic Binding Model with Complex Allostery. The depletion of free testosterone by increasing SHBG concentrations is best described by the new Multi-step Dynamic Binding Model with Complex Allostery. Fig. 5C depicts a graph of the heat of testosterone and SHBG association measured by isothermal calorimetry. The integrated ITC curve was generated after subtracting the buffer heats. SHBG starting concentration is 5 μΜ. Experimental points are shown by (■), and fit of the data to the new Multi-step Dynamic Binding Model with Complex Allostery model is shown by a solid line.
[0025] Fig. 6 depicts schematic representations of the various models tested in this study to examine SHBG:T interaction. Model A. Vermeulen's model, homogenous interaction of testosterone molecule with equal affinity for each monomer in SHBG dimer (Kd=lnM); Model B. monomers within the SHBG dimer exhibit distinct affinity constants with Kdi=lnM and Kd2 allowed to vary for data fits; Model C. Inter-subunit allostery with positive cooperativity for binding of two ligands such that Kd2=1nM, Kd1 allowed to vary and Kd2<Kd1; Model D. allostery with negative cooperativity for binding of two ligands such that Kd1=1nM, Kd2allowed to vary and Kd1<Kd2; and, Model E. The new Multi-step Dynamic Binding Model with Complex Allostery which encompasses two distinct SHBG microstates in equilibrium such that the equilibria between the unliganded and mono-liganded states readjust as testosterone concentration is increased.
Ravi Jasuja, Shalender Bhasin and Mikhail N Zakharov.
Hoping people understand the significance/importance of this testing method and the huge impact it will have.
Very long read but deeply interesting to say the least WO2014138026 METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS
1. (WO2014138026) METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS
Pub. No.: | WO/2014/138026 | International Application No.: | PCT/US2014/020223 |
Publication Date: | 12.09.2014 | International Filing Date: | 04.03.2014 |
IPC:
G01N 33/566 (2006.01) |
Applicants:
FUNCTION PROMOTING THERAPIES, LLC [US/US]; 32 Highland Meadows Lane Weston, Massachusetts 02493, US |
Inventors:
JASUJA, Ravi; US |
BHASIN, Shalender; US |
ZAKHAROV, Mikhail N.; US |
Agent:
CHEN, Yahua; Wolf, Greenfield and Sacks, P.C. 600 Atlantic Avenue Boston, MA 02210, US |
Priority Data:
61/772,054 | 04.03.2013 | US |
Title(EN)METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS
(FR)PROCÉDÉS ET SYSTÈMES POUR LE DIAGNOSTIC ET LE TRAITEMENT DE TROUBLES ANDROGÉNIQUES
Abstract:
(EN) The technology described herein is directed to the diagnosis and treatment of androgen disorders and/or deficiencies, e.g, low testosterone. |
SUMMARY
[0005] Described herein is the inventors' demonstration that the prevailing model of testosterone's binding to SHBG, used in the current methods of testing and diagnosis is flawed, and their further discovery of an improved model that permits methods, assays, and systems with greater accuracy and reliability in measuring testosterone levels. The multi-step dynamic binding model with complex allostery described herein is a new model for calculation of free testosterone. The multi-step dynamic binding model with complex allostery model is a modified ensemble allostery model that takes into consideration the specific SHBG-Testosterone binding interaction described herein.
[0006] In one aspect, described herein is a computer implemented method for an assay, comprising: on a device having one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs including instructions for: a) receiving data from measuring i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration in a biological sample obtained from an individual, to determine free testosterone concentration from the individual; b) attributing at least two distinct interconverting microstates of an unliganded SHBG dimer having a first monomer and a second monomer; and c) calculating the free testosterone concentration in the individual using the New Multi-Step Dynamic Binding Model with Complex Allostery encompassing readjustment of a first equilibria between the microstates upon binding of a first testosterone molecule to the first monomer and an allosteric interaction between two binding sites of the SHBG dimer.
*Refer to article to read [0007] - [0017]
[0018] In one aspect, described herein is a method for determining a need for adjustment of a dose of testosterone administered to an individual comprising a) determining the concentration of free testosterone in an individual receiving testosterone therapy at a first dose, wherein the concentration of free testosterone is determined by b) measuring i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration in a biological sample obtained from an individual, to determine free testosterone concentration from the individual; c) attributing at least two distinct interconverting microstates of an unliganded SHBG dimer having a first monomer and a second monomer by applying the New Multi-Step Dynamic Binding Model with Complex Allostery to the data of steps a) and b); d) calculating the free testosterone concentration in the individual using the New Multi-step Dynamic Binding Model with Complex Allostery encompassing readjustment of a first equilibria between the microstates upon binding of a first testosterone molecule to the first monomer and an allosteric interaction between two binding sites of the SHBG dimer; e) providing a second dose of testosterone that is higher than the first dose when the free testosterone concentration is below the lower end of the target therapeutic range (e.g.164 pg/ml); and f) providing a second dose of testosterone that is lower than the first dose when the free testosterone concentration is above the upper end of the target therapeutic range (314 pg/ml).
[0019] In some embodiments of any of the foregoing aspects, the step of attributing can be performed according to Figures 2, 3, 5, and 7. In some embodiments, of any of the foregoing aspects, the step of calculating can be performed according to Figure 7 or Example 5. In some embodiments of any of the foregoing aspects, the individual is a male over the age of 35. In some embodiments of any of the foregoing aspects, the androgen disorder is selected from the group consisting of a testosterone deficiency, an androgen deficiency, a hyperandrogenic disorder, an androgen expressing tumor, and a hypogonadism disorder. In some embodiments of any of the foregoing aspects, the androgen disorder is a hyperandrogenic disorder selected from the group consisting of an acne disorder, a hirsutism disorder, and an androgenic alopecia disorder. In some embodiments of any of the foregoing aspects, the individual has been diagnosed with a disease selected from the group consisting of: diabetes, human immunodeficiency virus (HIV), hepatitis B, hepatitis C, hypothyroidism or hyperthyroidism, androgen insensitivity, acromegaly, anorexia, muscular dystrophy, liver disease, cancer cachexia, malnutrition, nephrotic syndrome, and obesity, and other conditions in which SHBG or albumin concentrations are altered. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of classifying the individual into categories based on additional clinical symptoms. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of using the free testosterone concentration determined using the new Multistep Dynamic Binding Model with Complex Allostery to determine the dose or to individually adjust the dose of a formulation of testosterone for the treatment of a medical disease, taking into account patient's age, body weight and body mass index, medical conditions, including any co-morbid conditions, albumin and SHBG, and/or LH and FSH concentrations, and other patient-specific factors. In some embodiments of any of the foregoing aspects, instead of steps a-c, the data received is a previously calculated concentration of free testostosterone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figs. 1A-1B demonstrate that free T values calculated using the Vermeulen/ Sodergard/ Mazer model - the commonly used method for determining free T concentrations - differ from those measured using equilibrium dialysis. Fig. 1A depicts a graph of free testosterone concentrations in samples derived from a randomized testosterone trial (The TED study) were measured using the equilibrium dialysis and plotted against those calculated using the Vermeulen/ Sodergard/ Mazer equation. Calculated free T concentrations differed systematically from the measured values. Fig. 1B depicts a Bland Altman plot revealing substantial discrepancy between the calculated and measured free T concentrations.
[0021] Figs. 2A-2C demonstrate that binding of testosterone to SHBG displays complex behavior. Fig. 2A depicts a graph demonstrating that binding isotherms display complex behavior. Graded concentrations of testosterone were incubated overnight with various SHBG concentrations and the amount of bound testosterone was plotted against total T (added) concentrations (squares) 20nM, (triangles) 10 nM, (circles) 5nM SHBG. The fit curves represent the result of the linked fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery (Fig. 3). Fig. 2B depicts a graph of the depletion of free testosterone by varying SHBG concentration which is best described by the new Multi-step Dynamic Binding Model with Complex Allostery. Shown is concentration of free testosterone in the equilibrium dialysis experiment. One side of the equilibrium dialysis membrane has varying concentration of SHBG in buffer, the other one has plain buffer. Constant concentration of testosterone is added to each well of multi-well dialyzer: (triangles) 8.7nM. Curves are the result of the linked fit of data in Panel B to the new Multi-step Dynamic Binding Model with Complex Allostery (Fig 3).
Fig. 2C depicts a graph of the heat of T:SHBG association measured by isothermal calorimetry (ITC). Presented is the integrated ITC curve with buffer heats subtracted. SHBG starting concentration is 5μΜ. Experimental points are shown by squares, fit to the model in Fig.3 is shown by a solid line.
[0022] Fig. 3 depicts a schematic representation of the new Multi-step Dynamic Binding Model with Complex Allostery of testosterone's binding to SHBG and albumin, developed in this study. Unliganded SHBG dimers (S2, S2') exist in conformational equilibrium. Upon binding of the first testosterone molecule to microstates S2 and S2' can result in conformationally heterogeneous intermediate states S2T and S2'T respectively. These singly-occupied microstates then converge to S2T2 upon binding of the second testosterone molecule.
[0023] Figs. 4A-4B demonstrate a comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation (23) as implemented by Mazer (24) or the New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery with those measured using the equilibrium dialysis in samples from the 5alpha reductase trial. Fig. 4A depicts a graph of a comparison of the free testosterone concentration calculated by the Vermeulen equation and the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery to that measured by equilibrium dialysis in samples from a randomized testosterone trial in men. (■) free testosterone concentrations derived using an algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery; (•) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24). Solid lines are lines of best linear fit. Regression lines fit new Multi-step Dynamic Binding Model with Complex Allostery calculation (slope = 1.01±0.01, regression line fitting the squares), and the Vermeulen model (slope 0.77±0.02, lower line fitting the dots). Magenta dashed line is the line of prefect correlation. Fig. 4B depicts Bland Altman plots of the relative frequency distribution of % difference of calculated and measured free testosterone using either the Vermeulen equation (squares) or the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery (black dots) The relative deviations from the measured value are distributed around 0 for new Multi-step Dynamic Binding Model with Complex Allostery model and are different from zero for the Vermeulen model.
Figs. 4C-4D depict a Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation or the New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery with those measured using the equilibrium dialysis. Figs. 4C-4D demonstrate a Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation or the New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery with those measured using the equilibrium dialysis in samples from a randomized testosterone trial in men with ED. Fig. 4C depicts a graph of a comparison of the free testosterone concentration calculated by the Vermeulen equation and the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery to that measured by equilibrium dialysis in samples from a randomized testosterone trial in men. (squares) free testosterone concentrations derived using an algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery; (circles) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24). Black regression line fits new Multi-step Dynamic Binding Model with Complex Allostery model calculation (slope = 1.01±0.01 is shown) Fig. 4D depicts Bland Altman plots of the relative frequency of % difference of calculated and measured free testosterone using either the Vermeulen equation or the new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery. The relative deviations from the measured value are distributed around 0 for the new Multi-step Dynamic Binding Model with Complex Allostery model and are different from zero for the Vermeulen model.
[0024] Fig. 5A depicts a graph of the binding isotherm. Graded concentrations of testosterone were incubated overnight with 20 nM SHBG and the amount of bound testosterone was plotted against total testosterone concentration. The fit curve represents the fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery. Fig. 5B depicts a graph of the depletion curve. A constant concentration of testosterone (8.7 nM) was incubated with increasing SHBG concentrations. Free testosterone concentration is plotted against SHBG concentration. Solid line represents the fit of data to new Multi-step Dynamic Binding Model with Complex Allostery. The depletion of free testosterone by increasing SHBG concentrations is best described by the new Multi-step Dynamic Binding Model with Complex Allostery. Fig. 5C depicts a graph of the heat of testosterone and SHBG association measured by isothermal calorimetry. The integrated ITC curve was generated after subtracting the buffer heats. SHBG starting concentration is 5 μΜ. Experimental points are shown by (■), and fit of the data to the new Multi-step Dynamic Binding Model with Complex Allostery model is shown by a solid line.
[0025] Fig. 6 depicts schematic representations of the various models tested in this study to examine SHBG:T interaction. Model A. Vermeulen's model, homogenous interaction of testosterone molecule with equal affinity for each monomer in SHBG dimer (Kd=lnM); Model B. monomers within the SHBG dimer exhibit distinct affinity constants with Kdi=lnM and Kd2 allowed to vary for data fits; Model C. Inter-subunit allostery with positive cooperativity for binding of two ligands such that Kd2=1nM, Kd1 allowed to vary and Kd2<Kd1; Model D. allostery with negative cooperativity for binding of two ligands such that Kd1=1nM, Kd2allowed to vary and Kd1<Kd2; and, Model E. The new Multi-step Dynamic Binding Model with Complex Allostery which encompasses two distinct SHBG microstates in equilibrium such that the equilibria between the unliganded and mono-liganded states readjust as testosterone concentration is increased.
Last edited:
madman
Super Moderator
[0026] Fig. 7 depicts the thermodynamic Parameters associated with testosterone's binding to SHBG derived from the fit of binding isotherms and ITC data to new model. While this parameter set is not unique, together they consistently describe the binding isotherms, depletion curves and ITC data to the new Multi-step Dynamic Binding Model with Complex Allostery developed in this study. These were utilized to obtain FT values (cFTZBJ) in samples obtained in clinical trials.
[0027] Figs. 8A-8B demonstrate that the binding of testosterone to SHBG displays complex allostery. Fig. 8A depicts a graph demonstrating that binding isotherms display significant non-linearity. Varying concentrations of testosterone were incubated with a fixed concentration of SHBG (5, 10 or 20 nM) and bound testosterone was plotted against total testosterone concentration. The binding isotherms were generated at 5, 10 and 20 nM SHBG. Curves represent the result of the fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery.
Fig. 8B depicts a graph demonstrating that depletion of FT by varying SHBG concentration is best described by the new Multi-step Dynamic Binding Model with Complex Allostery. Constant concentration of testosterone (6, 12 , 17 or 32 nM) was incubated with increasing SHBG concentrations, and free testosterone concentration in buffer side was plotted against SHBG concentration. The curves are the result of the fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery.
[0028] Figs. 9A-9C depicts graphs depicting the fits of the various models of testosterone's binding to SHBG to the experimental data from binding isotherms, depletion experiments, and ITC. Left panels: The figures show the fits of data to the various models examined in this study.Right panels: The figures show corresponding residuals of the fit of data to various models of testosterone's binding to SHBG. Neither the Vermeulen's equation nor the simple allostery models adequately fit the experimental data from binding isotherms, depletion experiments, or ITC. The new Multi-step Dynamic Binding Model with Complex Allostery (model E) provided the optimal fit to the experimental data from all three methods.
[0029] Fig. 10 depicts a schematic of the control of testosterone levels.
[0030] Fig. 11 depicts a schematic of an exemplary system of determining free testosterone levels and/or dosages.
[0031] Fig. 12 depicts a device or a computer system 1000 comprising one or more processors 1300 and a memory 1500 storing one or more programs 1600 for execution by the one or more processors 1300.
Last edited:
madman
Super Moderator
We would need to keep this in mind.
Assessment of free testosterone concentration (2019)
Brian G Keevil, Jo Adaway
Highlights
Free testosterone may complement the measurement of total serum T, but this view remains controversial and is not universally accepted
Equilibrium dialysis methods are too complex for routine clinical use
Equations for calculating free testosterone are inaccurate because they were founded on faulty models of T binding to SHBG
The free androgen index is not recommended for use in males or females because of inaccuracy when the SHBG concentration is low.
More accurate equations are needed to calculate free testosterone, based on more detailed knowledge of the binding of testosterone to SHBG.
Harmonization of T and SHBG assays between laboratories needs to be improved
*The algorithms derived for calculating free T are inaccurate because they were founded on faulty models of testosterone binding to SHBG
*the effects of differences in binding protein constants
The calculation proposed by Vermeulen et al has been the most widely used but the main criticism of all these equations is that the model of binding of T to SHBG may not be accurate, and in addition the set of binding constants used may not be appropriate (14, 32). SHBG is a homodimeric glycoprotein with a molecular weight of approximately 90 kDa (33) and the distribution of testosterone bound to SHBG is different in males and females. When Estradiol is present, approximately 20% of SHBG binding sites are occupied by testosterone (34). It was previously thought that the two binding sites on the SHBG molecule are equivalent, but using modern biophysical techniques it is now known that the binding sites are not equivalent, and they each bind SHBG with a different affinity (22). As a result of this, alternative models of binding of T to SHBG have been advocated (14,22).
Calculated FT (cFT) estimates are based on the assumption that there is normal steady-state protein binding for T and the equations are dependent on reliability and accuracy of both the T and SHBG assays (6, 35, 36). All of the equations will estimate free T incorrectly when the protein concentrations differ widely from physiological values, if there are large concentrations of competing steroids, or if the SHBG binding affinity is affected by a rare genetic variant (12, 37). Depending on the equation used, systematic differences between free T estimates have been reported compared with measured free T (18, 21, 30). Discrepancies between free T measured using equilibrium dialysis and cFT are most likely caused by erroneous modelling of testosterone binding to SHBG.
Recent work shows that cFT-Vermeulen is strongly correlated to free T measured by the reference method (direct equilibrium dialysis), but free T is overestimated by 20 to 30%, thus agreeing with previous work (21, 38, 39). However, the relationship between cFT-Vermeulen and measured free T was found to be linear and independent of serum T, albumin and SHBG concentrations. The lack of reliance on SHBG in the Vermeulen equation was thought to be strength of the cFT-Vermeulen since assessment of free T is especially important in patients at the extremes of the SHBG concentration range. The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
Equations for calculating FT are inaccurate because they were founded on faulty models of T binding to SHBG. Calculated FT methods offer are simple and inexpensive and may offer the best way forward, but more accurate equations are needed and these must be based on more detailed knowledge of the complicated binding testosterone to SHBG.
Assessment of free testosterone concentration (2019)
Brian G Keevil, Jo Adaway
Highlights
Free testosterone may complement the measurement of total serum T, but this view remains controversial and is not universally accepted
Equilibrium dialysis methods are too complex for routine clinical use
Equations for calculating free testosterone are inaccurate because they were founded on faulty models of T binding to SHBG
The free androgen index is not recommended for use in males or females because of inaccuracy when the SHBG concentration is low.
More accurate equations are needed to calculate free testosterone, based on more detailed knowledge of the binding of testosterone to SHBG.
Harmonization of T and SHBG assays between laboratories needs to be improved
*The algorithms derived for calculating free T are inaccurate because they were founded on faulty models of testosterone binding to SHBG
*the effects of differences in binding protein constants
The calculation proposed by Vermeulen et al has been the most widely used but the main criticism of all these equations is that the model of binding of T to SHBG may not be accurate, and in addition the set of binding constants used may not be appropriate (14, 32). SHBG is a homodimeric glycoprotein with a molecular weight of approximately 90 kDa (33) and the distribution of testosterone bound to SHBG is different in males and females. When Estradiol is present, approximately 20% of SHBG binding sites are occupied by testosterone (34). It was previously thought that the two binding sites on the SHBG molecule are equivalent, but using modern biophysical techniques it is now known that the binding sites are not equivalent, and they each bind SHBG with a different affinity (22). As a result of this, alternative models of binding of T to SHBG have been advocated (14,22).
Calculated FT (cFT) estimates are based on the assumption that there is normal steady-state protein binding for T and the equations are dependent on reliability and accuracy of both the T and SHBG assays (6, 35, 36). All of the equations will estimate free T incorrectly when the protein concentrations differ widely from physiological values, if there are large concentrations of competing steroids, or if the SHBG binding affinity is affected by a rare genetic variant (12, 37). Depending on the equation used, systematic differences between free T estimates have been reported compared with measured free T (18, 21, 30). Discrepancies between free T measured using equilibrium dialysis and cFT are most likely caused by erroneous modelling of testosterone binding to SHBG.
Recent work shows that cFT-Vermeulen is strongly correlated to free T measured by the reference method (direct equilibrium dialysis), but free T is overestimated by 20 to 30%, thus agreeing with previous work (21, 38, 39). However, the relationship between cFT-Vermeulen and measured free T was found to be linear and independent of serum T, albumin and SHBG concentrations. The lack of reliance on SHBG in the Vermeulen equation was thought to be strength of the cFT-Vermeulen since assessment of free T is especially important in patients at the extremes of the SHBG concentration range. The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
Equations for calculating FT are inaccurate because they were founded on faulty models of T binding to SHBG. Calculated FT methods offer are simple and inexpensive and may offer the best way forward, but more accurate equations are needed and these must be based on more detailed knowledge of the complicated binding testosterone to SHBG.
Last edited:
madman
Super Moderator
*However, the relationship between cFT-Vermeulen and measured free T was found to be linear and independent of serum T, albumin and SHBG concentrations.
*The lack of reliance on SHBG in the Vermeulen equation was thought to be strength of the cFT-Vermeulen since assessment of free T is especially important in patients at the extremes of the SHBG concentration range.
*The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
*The lack of reliance on SHBG in the Vermeulen equation was thought to be strength of the cFT-Vermeulen since assessment of free T is especially important in patients at the extremes of the SHBG concentration range.
*The bias is probably due to imperfect estimations of the association constants for the binding of T to SHBG and albumin, as discussed above, and this would need to be allowed for when comparing different methods for cFT.
madman
Super Moderator
Now does everyone truly know where their free testosterone levels sit and how many are experiencing negative effects/sides on said protocol only to end up chasing their tale from unknowingly having too high FT levels.
Assessment of free testosterone concentration (2019)
Brian G Keevil, Jo Adaway
*The older, direct analogue RIA methods have been discredited and are no longer recommended for use (6-8).
Assessment of free testosterone concentration (2019)
Brian G Keevil, Jo Adaway
*The older, direct analogue RIA methods have been discredited and are no longer recommended for use (6-8).
Deleted member 16042
Member
Mind blown. I am utterly confused.
I plugged my numbers into TruT from a set of labs:
1115 TT ng/dl
SHBG 50 nMol/L
Albumin 4.7 g/dl
and it calculated Free Testosterone: 1.22 nMol/L, 35.16 ng/dL.
When converted that's 351.6 pg/ml compared to direct at 18.8 pg/ml
So are we looking at an entire revamp of the direct assay pg/ml reference? Different method, utterly different range?
I plugged my numbers into TruT from a set of labs:
1115 TT ng/dl
SHBG 50 nMol/L
Albumin 4.7 g/dl
and it calculated Free Testosterone: 1.22 nMol/L, 35.16 ng/dL.
When converted that's 351.6 pg/ml compared to direct at 18.8 pg/ml
So are we looking at an entire revamp of the direct assay pg/ml reference? Different method, utterly different range?
The direct free T assays should not be used, period. Not only are they on an entirely different scale, by an order of magnitude, but they also do not correlate well with either equilibrium dialysis or ultrafiltration. In short they are just plain inaccurate. The TruT calculation correlates well with the high-quality tests and is on virtually the same scale. The Vermeulen free T calculation correlates well with the good tests, but is effectively on a lower scale.
Deleted member 16042
Member
Understood, I get that, so wondering if my good doctor (Saya) is dropping free T blood work in favor of TruT calcs and adopting 164 to 314 pg/ml reference range?
Deleted member 16042
Member
Yeah I know, I've been with Saya for a couple years, but with this new info I wonder if his perspective will change.
It would actually be helpful to me to drop free T direct testing in favor of CMP which has albumin as I'd like to monitor liver due to taking green tea extract. Just would be nice to trade off costs. I know CMP is only $15, but cumulative costs add up in a big way for me.
Same.
New info is huge. I was rockin T in the mid 700s but got told since my free t direct was 8 that trt would help me. Shbg was in the 80s...at least that’s down to 45 now
With my original scenario my tru T was 20+, not 8...live and learn. Didn’t know about the different free T test accuracy issues back then.
New info is huge. I was rockin T in the mid 700s but got told since my free t direct was 8 that trt would help me. Shbg was in the 80s...at least that’s down to 45 now
With my original scenario my tru T was 20+, not 8...live and learn. Didn’t know about the different free T test accuracy issues back then.
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