Free Hormone Hypothesis

[Michael Scally MD]

Essential Points

· Most of the circulating testosterone is bound to its cognate binding proteins – sex hormone binding globulin (SHBG), human serum albumin (HSA), cortisol binding globulin, and orosomucoid. These binding proteins play an important role in regulating the transport, tissue delivery, bioactivity, and metabolism of testosterone.

· The physiochemical characteristics and dynamics of testosterone’s binding to its binding proteins are poorly understood. Over-simplified assumptions of stoichiometry, binding dynamics, and binding affinity have contributed to the development of inaccurate linear binding models of testosterone binding to SHBG and HSA

· The ensemble allosteric model of testosterone’s binding to SHBG developed from recent studies using modern biophysical techniques suggests that testosterone binding to SHBG is a complex, multistep process that involves inter-binding site allostery.

· The dynamics of testosterone’s binding to HSA, orosomucoid and CBG also require careful reexamination as the roles of these binding proteins in regulating circulating testosterone concentrations remain incompletely understood.

· If the free hormone hypothesis is correct, i.e. only the free testosterone is biologically active, accurate determination and harmonized reference ranges for free testosterone are necessary to diagnose androgen disorders in men and women.

· The methods for the measurement of free testosterone are fraught with potential problems including poor precision, inaccuracy, and specificity, and reliable assays are not readily available to practicing clinicians. Therefore, algorithms based on valid binding models that can be used to estimate the circulating free testosterone levels are needed to facilitate sound clinical decision making.

Goldman AL, Bhasin S, Wu FCW, Krishna M, Matsumoto AM, Jasuja R. A Reappraisal of Testosterone's Binding in Circulation: Physiological and Clinical Implications. Endocr Rev. Reappraisal of Testosterone's Binding in Circulation: Physiological and Clinical Implications | Endocrine Reviews | Oxford Academic

In circulation, testosterone and other sex hormones are bound to binding proteins which play an important role in regulating their transport, distribution, metabolism, and biologic activity. According to the free hormone hypothesis, which has been debated extensively, only the unbound or free fraction is biologically active in target tissues.

Consequently, accurate determination of the partitioning of testosterone between bound and free fractions is central to our understanding of how its delivery to the target tissues and biological activity are regulated, and, consequently, to the diagnosis and treatment of androgen disorders in men and women.

Here, we present an historical perspective of the evolution of our current understanding of testosterone's binding to circulating binding proteins. Based on an appraisal of the literature and new experimental data, we show that the assumptions of stoichiometry, binding dynamics, and binding affinity of the prevailing models of testosterone binding to sex hormone-binding globulin (SHBG) and human serum albumin (HSA) are not supported by the published experimental data and are likely inaccurate.

The review offers some guiding principles in the application of free testosterone measurements in the diagnosis and treatment of patients with androgen disorders. The growing number of testosterone prescriptions and the widely recognized problems with methods for the direct measurement as well as the computation of free testosterone concentrations render this critical review timely and clinically relevant.

 

[Michael Scally MD]

[OA] Handelsman DJ. Free Testosterone: Pumping up the Tires or Ending the Free Ride?. Endocrine Reviews. 2017;38(4):297–301. https://academic.oup.com/edrv/article/38/4/297/4071740/Free-Testosterone-Pumping-up-the-Tires-or-Ending

The review by Goldman et al. is a strong restatement of the free hormone hypothesis (FHH) with its contingent focus on measurement of so-called “free” testosterone (FT). Reappraisal of Testosterone’s Binding in Circulation: Physiological and Clinical Implications | Endocrine Reviews | Oxford Academic

The FHH has even been recently described as a “central dogma of endocrinology”, Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis, reminiscent of the hubris of Crick’s central dogma of molecular biology stated as a unidirectional information transfer from DNA to RNA to protein, which proved neither a dogma (an unassailable proposition) nor universally correct.

The review provides an outstandingly clear and encyclopedic summation of what is and is not known of the molecular mechanisms of testosterone binding to circulating binding proteins. On the other hand, its coverage of FT measurement and its interpretation are both more selective and tendentious.

Above all the review’s overall focus on means rather than ends, on how to do it rather than why, the review belies why the application of FT measurements remain contentious so that less expert readers may wonder what controversy there is that the authors hint at fleetingly.

 

[Michael Scally MD]

Assessment of Free Testosterone Concentration

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.

Testosterone (T) is strongly bound to sex hormone binding globulin and measurement of free T may be more appropriate than measuring total serum T, according to the free hormone theory.

This view remains controversial and it has its detractors who claim that little extra benefit is gained than simply measuring total T, but it is endorsed by recent clinical practice guidelines for investigation of androgen disorders in both men and women.

Free T measurement is very challenging. The gold standard equilibrium dialysis methods are too complex for use in routine clinical laboratories, assays are not harmonized and consequently there are no common reference intervals to aid result interpretation.

The algorithms derived for calculating free T are inaccurate because they were founded on faulty models of testosterone binding to SHBG, however they can still give clinically useful results.

To negate the effects of differences in binding protein constants, some equations for free T have been derived from accurate measurement of testosterone in large population studies, however a criticism is that the equations may not hold true in different patient populations.

The free androgen index is not recommended for use in men because of inaccuracy at extremes of SHBG concentration, and in women it can also give inaccurate results when SHBG concentrations are low.

If the free hormone hypothesis is to be believed, then calculated free testosterone may offer the best way forward but better equations are needed to improve accuracy and these should be derived from detailed knowledge of testosterone binding to SHBG.

There is still much work to be done to improve harmonization of T and SHBG assays between laboratories because these can have a profound effect on the equations used to calculate free testosterone.

Keevil BG, Adaway J. Assessment of free testosterone concentration. J Steroid Biochem Mol Biol 2019. https://www.sciencedirect.com/science/article/abs/pii/S0960076019301979?via=ihub

 

[Michael Scally MD]

[OA] The Free Hormone Hypothesis: When, Why, and How to Measure the Free Hormone Levels to Assess Vitamin D, Thyroid, Sex Hormone, and Cortisol Status

The free hormone hypothesis postulates that only the nonbound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert biologic effects.

In this review, I will examine four hormone groups-vitamin D metabolites (especially 25OHD), thyroid hormones (especially thyroxine [T4]), sex steroids (especially testosterone), and glucocorticoids (especially cortisol)-that are bound to various degrees to their respective binding proteins-vitamin D-binding protein (DBP), thyroid-binding globulin (TBG), sex hormone-binding globulin (SHBG), and cortisol-binding globulin (CBG)-for which a strong case can be made that measurement of the free hormone level provides a better assessment of hormonal status than the measurement of total hormonal levels under conditions in which the binding proteins are affected in levels or affinities for the hormones to which they bind.

I will discuss the rationale for this argument based on the free hormone hypothesis, discuss potential exceptions to the free hormone hypothesis, and review functions of the binding proteins that may be independent of their transport role.

I will then review the complications involved with measuring the free hormone levels and the efforts to calculate those levels based on estimates of binding constants and levels of both total hormone and total binding protein.

In this review, the major focus will be on DBP and free 25OHD, but the parallels and differences with the other binding proteins and hormones will be highlighted. Vitamin D and its metabolites, thyroid hormones, sex steroids, and glucocorticoids are transported in blood bound to serum proteins.

The tightness of binding varies depending on the hormone and the binding protein such that the percent free varies from 0.03% for T4 and 25OHD to 4% for cortisol with testosterone at 2%. Although the major function of the primary carrier proteins (DBP, TBG, SHBG, and CBG) may be to transport their respective lipophilic hormones within the aqueous media that is plasma, these proteins may have other functions independent of their transport function.

For most tissues, these hormones enter the cell as the free hormone presumably by diffusion (the free hormone hypothesis), although a few tissues such as the kidney and reproductive tissues express megalin/cubilin enabling by endocytosis protein-bound hormone to enter the cell.

Measuring the free levels of these protein-bound hormones is likely to provide a better measure of the true hormone status than measuring the total levels in situations where the levels and/or affinities of the binding proteins are altered.

Methods to measure free hormone levels are problematic as the free levels can be quite low, the methods require separation of bound and free that could disturb the steady state, and the means of separating bound and free are prone to error.

Calculation of free levels using existing data for association constants between the hormone and its binding protein are likewise prone to error because of assumptions of linear binding models and invariant association constants, both of which are invalid.

Bikle DD. The Free Hormone Hypothesis: When, Why, and How to Measure the Free Hormone Levels to Assess Vitamin D, Thyroid, Sex Hormone, and Cortisol Status. JBMR Plus. 2020 Nov 2;5(1):e10418. doi: 10.1002/jbm4.10418. PMID: 33553985; PMCID: PMC7839820. https://asbmr.onlinelibrary.wiley.com/doi/10.1002/jbm4.10418

 

[Deleted member 59947]

First study says SHBG is important to shuttle hormones to their receptors.

Last study says SHBG prevents hormones from exerting their action.

So, which one is it?

 

[SUGARBEAR]

Albumin transports free t to receptors . shbg holds bound t for later use. make sense. shbg even above range may be effetive to prolong halflife