Hope for an accurate method to determine Free Testosterone

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madman

Super Moderator
Novel Algorithm for Free testosterone Determination



ABSTRACT

- the current methods for the determination of free testosterone- equilibrium dialysis, ultrafiltration, tracer analog methods, and the use of algorithms based on the LAW-OF-MASS-ACTION- suffer from problems of imprecision or inaccuracy

- equilibrium dialysis method, widely considered the reference method, is labor-intensive and cumbersome, and susceptible to errors due to tracer impurities, leading the Endocrine Society's Expert Panel to conclude that " the calculation of free testosterone is the most useful estimate of free testosterone in plasma"

- therefore, algorithms for calculating free testosterone concentrations from total testosterone, SHBG and albumin concentrations using the law-of-mass-action equations have been used widely

- our preliminary data show that the current model of homogenous testosterone binding sites on SHBG- the conceptual basis of the extent law-of-mass-action equations- is erroneous, and that the free testosterone concentrations derived from the available law-of-mass-action equations differ substantially from those measured by equilibrium dialysis

- because of these limitations of the available methods, there is an enormous unmet need for an accurate method to determine free testosterone concentrations that can be applied conveniently in clinical practice

- new evidence shows that testosterone's binding to SHBG conforms to an Ensemble Allostery Model (EAM)

- based on this new EAM model of testosterone binding to SHBG, we have constructed a novel algorithm for calculating free testosterone levels that provide excellent conformity with values determined by equilibrium dialysis

- this Phase I SBIR application aims to characterize the technical performance of the novel algorithm by determining its precision, accuracy, functional sensitivity, intra-and inter-assay variation, and specifity, stability, and matrix effect (AIM 1)



AIM 2- will incorporate SHB genotype in the algorithm and apply it to men with different SHBG genotypes

AIM 3- will characterize the performance of the assay in young and older men and women, and in obese men with alterations in SHBG concentrations



Project Start 2014-09-15
Project End 2015-08-31



http://grantome.com/grant/NIH/R43-AG045011-01A1
 
Last edited:
Defy Medical TRT clinic doctor

madman

Super Moderator
Phase II: Research and Commercialization of TruT Algorithm for Free Testosterone



ABSTRACT

- the measurement of testosterone(T) levels is central to the diagnosis of androgen disorders, such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women

- circulating t is bound with high affinity to sex hormone binding globulin (SHBG) and with substantially lower affinity to albumin; only the free fraction is biologically active

- conditions that affect SHBG concentrations, such as aging and obesity, alter total T but not free T concentrations; in these conditions, the determination of free t is necessary to obtain an accurate assessment of androgen status

- tracer analog method, the most widely used method for free T, has been shown to be inaccurate

- equilibrium dialysis method is, technically difficult to implement and standardize, and is not available in most hospital laboratories, leading the Endocrine Society's Expert Panel to conclude that?? the calculation of free testosterone is the most useful estimate of free testosterone in plasma??

- therefore, there is an unmet need for algorithms that provide accurate estimates of free T that match those derived from equilibrium dialysis

- we have designed a novel and accurate TruTTM algorithm for the determination of free T, based on the characterization of testosterones's binding to SHBG using modern biophysical techniques

- we have discovered that testosterone's binding to SHBG is a dynamic multistep process that includes allosteric interaction between the two binding sites on an SHBG dimer

- our computational frame-work incorporates the correct binding parameters derived experimentally in these studies, the non-linear dynamics in T:SHBG association, and allsotery

- in phase I studies , we demonstrated that TruTTM algorithm provides accurate free T values that match those obtained using the equilibrium dialysis in healthy and hypogonadal men

- we have also shown that the binding parameters that have formed the basis of previous equations (e.g., Vermeulen) are incorrect, and that free T values derived using these equations deviate substantially from free T measured by equilibrium dialysis

- the phase I studies have led to adoption of the TruTTM algorithm at several institutions

- the phase II program will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentration, such as obesity and aging (AIM 1), in healthy women across the menstrual cycle, and in women with PCOS (AIM 2)

- we will generate population-based reference ranges for free T (AIM 3)

- phase II also includes plans for commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption

-the phase II program will provide validation of TruTTM algorithm in the two most common clinical indications for free T measurement? men suspected of hypogonadism and altered SHBG levels, and women with hyperandrogenic disorders

- it will also enable the development of a HIPAA compliant platform that can be embedded into electronic medical record for wider clinical adoption and for improving clinical care



Project Start 2014-09-15
Project End 2019-05-31



http://grantome.com/grant/NIH/R44-AG045011-02
 
Last edited:

Blackhawk

Member
Hope this is not too much a tangent. Just the assumption: "only the free fraction is biologically active" is not completely true. While it still remains especially very important in the context of low SHBG, there may be newer thinking coming on this

"only the free fraction is biologically active"

This is apparently false when it comes to sex hormone sensitive tissues including prostate, breast and endometrium. It wasn't stated comprehensively which tissues are affected, but it is likely to be more, like potentially testicle cells, ovary etc. See Nick Sakas video with Jay Campbell: https://www.youtube.com/watch?v=a9sbWHAmVrA

I took notes. still have to go back and proof them based on what Sakas says, but here's a rough summary:

SHBG: proteins produced in liver bind T and estrogens.

Estimated 2-3% of sex hormones unbound.

1980's researchers used non hormone responsive cultured cells in vitro and added T E2 and SHBG. When adding SHBG, steroid hormones had no effect on the cells. They concluded that SHBG made sex hormones ineffective.

Free hormone hypothesis based on this stated SHBG interferes with sex hormone uptake of cells. There was no other known benefit of SHBG.

However, in 2005 researchers proved that tissue specific receptors exist which SHBG binds to, then Sex hormones trigger downstream reactions/responses in the cells leading to gene expression inside the cell. This is mediated by a messenger molecule; CMP cyclic adenosine mono phosphate which can active specific Kinases and is also involved in regulation of adrenaline and glucagon.

This creates a nongenomic pathway of signaling gene and protein expression inside the cell.

In contrast,Testosterone attaching to cell androgen receptors is a genomic pathway.

The implication is in the case of these specific cell types, the T and E2/SHBG complexes are effective attaching to their specific receptors and affecting cellular process despite being bound to SHBG. This dispells some of the free T hypothesis.

Also found megalin a receptor of LDL protein family which sequesters vitamins A and D inside the cell, then the ligands are free after binding molecules are broken down so Vit A & D can exert their role in the cell. Megalin can also sequester the complex of SHBG and sex steroids. This is another genomic pathway of SHBG/sex hormone complex signaling inside the cell.

There is another implication from other researchers who spotted a receptor named chubalin(?) which does not actively internalize SHBG and sex hormones, it serves as a receptor for albumin which also binds to steroid hormones,

These things mean we may need to change how we assess SHBG levels in relationship to sex hormone metabolism.



I am going to try to get a list of the references he cites.
 

madman

Super Moderator
Hope this is not too much a tangent. Just the assumption: "only the free fraction is biologically active" is not completely true. While it still remains especially very important in the context of low SHBG, there may be newer thinking coming on this

"only the free fraction is biologically active"

This is apparently false when it comes to sex hormone sensitive tissues including prostate, breast and endometrium. It wasn't stated comprehensively which tissues are affected, but it is likely to be more, like potentially testicle cells, ovary etc. See Nick Sakas video with Jay Campbell: https://www.youtube.com/watch?v=a9sbWHAmVrA

I took notes. still have to go back and proof them based on what Sakas says, but here's a rough summary:

SHBG: proteins produced in liver bind T and estrogens.

Estimated 2-3% of sex hormones unbound.

1980's researchers used non hormone responsive cultured cells in vitro and added T E2 and SHBG. When adding SHBG, steroid hormones had no effect on the cells. They concluded that SHBG made sex hormones ineffective.

Free hormone hypothesis based on this stated SHBG interferes with sex hormone uptake of cells. There was no other known benefit of SHBG.

However, in 2005 researchers proved that tissue specific receptors exist which SHBG binds to, then Sex hormones trigger downstream reactions/responses in the cells leading to gene expression inside the cell. This is mediated by a messenger molecule; CMP cyclic adenosine mono phosphate which can active specific Kinases and is also involved in regulation of adrenaline and glucagon.

This creates a nongenomic pathway of signaling gene and protein expression inside the cell.

In contrast,Testosterone attaching to cell androgen receptors is a genomic pathway.

The implication is in the case of these specific cell types, the T and E2/SHBG complexes are effective attaching to their specific receptors and affecting cellular process despite being bound to SHBG. This dispells some of the free T hypothesis.

Also found megalin a receptor of LDL protein family which sequesters vitamins A and D inside the cell, then the ligands are free after binding molecules are broken down so Vit A & D can exert their role in the cell. Megalin can also sequester the complex of SHBG and sex steroids. This is another genomic pathway of SHBG/sex hormone complex signaling inside the cell.

There is another implication from other researchers who spotted a receptor named chubalin(?) which does not actively internalize SHBG and sex hormones, it serves as a receptor for albumin which also binds to steroid hormones,

These things mean we may need to change how we assess SHBG levels in relationship to sex hormone metabolism.



I am going to try to get a list of the references he cites.




Yes I already understand that, thanks https://www.excelmale.com/forum/sho...ation-Physiological-and-Clinical-Implications
 

madman

Super Moderator
The TruT algorithm looks promising.....time will tell as 2019 is the expected end date of the phase II project study.
 

madman

Super Moderator
More accurate measurements are great, but I'm not sure it would change how people are treated or should be. To me it's about health and negative symptoms/quality of life. If a dr could accurately nail down the correct protocol faster that would be great. Otherwise we still experiment until we feel great and health indicators are good.


Well said!
 

Kirk001

Member
So these free T calculators on the internet like http://www.issam.ch/freetesto.htm are not accurate?

So I think that calculator there is what is being referenced in madman's original post "...therefore, algorithms for calculating free testosterone concentrations from total testosterone, SHBG and albumin concentrations using the law-of-mass-action equations have been used widely" and are "erroneous."

Sounds like the direct measurement of free T (standard LabCorp type) is least accurate, and the the calculation method (based on SHBG, total T and albumin) is more accurate, but this new approach should be even more accurate.
 

madman

Super Moderator
This is the central problem. If the measurement (of total and free) is not precise then dosing becomes a trial and error. Either the person gets to supra-physiological or sub-therapeutic levels.

Contributors to the substantial variation in on-treatment testosterone levels in men receiving transdermal testosterone gels in randomized trials. - PubMed - NCBI ;
SHBG and therefore, Free T levels are important..


Of course accurate measurements of hormones are critical as SHBG/FT levels are of great importance to an effective trt protocol let alone overall health.

There is much more to SHBG than simply high/low levels.

Polymorphisms/mutants and some points noted below.





A Reappraisal of Testosterone’s Binding in Circulation: Physiological and Clinical Implications

Anna L. Goldman, Shalender Bhasin, Frederick C.W. Wu, Meenakshi Krishna, Alvin M. Matsumoto, and Ravi Jasuja



Multiple hypothetical mechanisms for the cellular uptake of testosterone and downstream signaling

(a) the model depicting the "free" hormone hypothesis

(b) the megalin dependent mode of testosterone entry

(c) the SHBG receptor-testosterone system

(d) steroid ligand-dependent interactions between SHBG and at least two matrix-associated proteins in the fibulin family


https://academic.oup.com/view-large/figure/94946825/er.2017-00025f4.tif












Naturally Occurring Mutants Inform SHBG Structure and Function
Tsung-Sheng Wu Geoffrey L. Hammond
 

Attachments

  • APR24-SHBG-MUTANTS-mend1026.pdf
    2.3 MB · Views: 76

madman

Super Moderator


*The binding of T to SHBG is complex, which results in many different methods that directly measure or calculate free T. Some of these methods do not measure the free fraction of T and some formulae may provide less accurate results [40]

*Recent evidence suggests that the law of mass action formula which is based on the assumption that two T molecules bind to two binding sites on the SHBG with similar binding affinity may be incorrect. And further argues that the binding of T to SHBG may be a multistep, dynamic process with complex allosteric characteristics [65]. Based on this new model, investigators used a new formula to calculate free T in younger men in the Framingham Heart Study and showed that the newly calculated values were similar to those measured by equilibrium dialysis. They further verified that the calculated free T values had clinical diagnostic validity using data from the European Male Aging Study

*Currently, the CDC is developing a harmonized method for free T based on calculated free T using revised formulae. This may bring the measurement of free T to a referable standard in clinical laboratories and common reference intervals that all clinicians can use


*Perhaps the newer formula for calculated free T validated in multiple laboratories [65], will become generally available, correlate with free T by equilibrium dialysis and demonstrate improved correlation with clinical symptoms and therapeutic responsiveness. If all these prove to be true, then this formula to calculate free T may be a justified replacement for free T measurement by the equilibrium dialysis methodology




Phase II: Research and Commercialization of TruT Algorithm for Free Testosterone
Jasuja, Ravi

https://grantome.com/grant/NIH/R44-AG045011-02


The measurement of testosterone (T) levels is central to the diagnosis of androgen disorders, such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women. Circulating T is bound with high affinity to sex hormone-binding globulin (SHBG) and with substantially lower affinity to albumin; only the free fraction is biologically active. Conditions that affect SHBG concentrations, such as aging and obesity, alter total but not free T concentrations; in these conditions, the determination of free T is necessary to obtain an accurate assessment of androgen status. The tracer analog method, the most widely used method for free T, has been shown to be inaccurate. The equilibrium dialysis method, considered the reference method, is technically difficult to implement and standardize, and is not available in most hospital laboratories, leading the Endocrine Society's Expert Panel to conclude that ?? the calculation of free testosterone is the most useful estimate of free testosterone in plasma?? Therefore, there is an unmet need for algorithms that provide accurate estimates of free T that match those derived from equilibrium dialysis. We have designed a novel and accurate TruTTM algorithm for the determination of free T, based on the characterization of testosterone's binding to SHBG using modern biophysical techniques. We have discovered that testosterone's binding to SHBG is a dynamic multistep process that includes allosteric interaction between the two binding sites on an SHBG dimer. Our computational framework incorporates the correct binding parameters derived experimentally in these studies, the non-linear dynamics in T: SHBG association, and allostery

In phase I studies, we demonstrated that the TruTTM algorithm provides accurate free T values that match those obtained using the equilibrium dialysis in healthy and hypogonadal men
. We have also shown that the binding parameters that have formed the basis of previous equations (e.g., Vermeulen) are incorrect, and that free T values derived using these equations deviate substantially from free T measured by equilibrium dialysis. The phase I studies have led to the adoption of the TruTTM algorithm at several institutions.

The phase II program will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentrations, such as obesity and aging (AIM 1), in healthy women across the menstrual cycle, and in women with PCOS (Aim 2).
We will generate population-based reference ranges for free T (Aim 3). Phase II also includes plans for the commercialization of the TruTTM algorithm using a HIPAA-compliant infrastructure for its clinical adoption

The phase II program will provide validation of the TruTTM algorithm in the two most common clinical indications for free T measurement? men suspected of hypogonadism and altered SHBG levels, and women with hyperandrogenic disorders. It will also enable the development of a HIPAA-compliant platform that can be embedded into the electronic medical record for wider clinical adoption and for improving clinical care
 
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