I want to preface this material by stating that the foregoing information should rock the TRT and medical communities. This information exists in studies, but I have never seen it presented to the masses in a fastidious, analytical, and cogent manner. This information completely dismantles the paradigm under which the medical community understands testosterone and testosterone therapy. This proves wrong the way the medical institution has educated physicians; this proves wrong the way the medical institution has educated those seeking treatment for low testosterone; this proves wrong the way the medical institution has educated the world. I don’t believe this information will have the effect that it should for a myriad of reasons, but mainly because it is common knowledge that the medical community is ignorant with regards to testosterone, and consequently, it is not that surprising.
The free hormone hypothesis is espoused by the endocrine society, and by extension, the medical community as a whole. The free hormone hypothesis assumes that only free testosterone is diffused into cells, where the free hormone binds to androgen receptors, subsequently translocates into the nucleus, binds to a promoter on DNA, and elicits a cascade of effects. According to the free hormone hypothesis, biological activity of testosterone is best reflected by free rather than total testosterone concentrations. In this model, testosterone bound SHBG is ignored. It may be relevant and worth noting that hypothesis is defined as “educated guess”. I am not sure the free hormone hypothesis fits the definition of hypothesis considering it is not very educated. Of interest, those of us with a vast amount of aggregated data in the field have noticed over time that we are more informed about a person’s sense of well-being – feeling optimal – from their total testosterone number, not their free testosterone. From a plethora of experience, people with lower end total T, and mid to high range free T, even though they may have functional erection quality, tend to report symptoms of hypogonadism. As expected, people with mid to upper range total T, high SHBG, and lower end free T, also present symptoms of hypogonadism; indicating that free T is an important metric, but not the most important metric. Surprisingly, upper range total T, upper range free T, and lower range SHBG may present with a sub-optimal sense of well-being that is unrelated to estrogenic side effects. Over time, I developed heuristics, a methodology, based on the teleological evidence of the best means to optimal results, which has included paying more attention to total T and SHBG than any free T metric. I knew this was the best practice, again, because it works very well, but I didn’t know exactly why until undertaking a deep dive into understanding the massive limitations of the free hormone hypothesis.
The free hormone hypothesis was conceptualized from experimental studies performed in the 1950’s to the 1980’s. What is interesting, is that many of these studies indicated that there was an incomplete understanding of the role of all binding proteins, and that there may be evidence that they play a more crucial biophysical and foundational role. Despite the assumptions and unknowns, the medical community espoused a facile and oversimplified free hormone hypothesis to anchor its paradigm under which testosterone and related hormones are understood. Additionally, the facile and oversimplified free hormone hypothesis would be used to mischaracterize, mistreat, and misdiagnose hormone deficiency for the next 5 decades and counting.
CBG and Orosomucoid:
Everyone understands that testosterone binds to Albumin and SHBG, but what is less known is that testosterone also binds to two other binding proteins: Cortico-steroid Binding Globulin (CBG) and orosomucoid. Early theory indicated that CBG bound to about 4% of circulating testosterone with low association, such that the binding of testosterone to CBG has been ignored. In follow-up in vitro studies that investigated the influence of other steroids on protein-bound testosterone, the experimentally observed binding of CBG to free testosterone in the addition of cortisol was 15.4%. This discordance between the predicted and experimentally obtained values raises the possibility that published estimates of testosterone binding to CBG are incorrect and that a higher amount of circulating testosterone is bound to CBG than was assumed. orosomucoid acts as a carrier of positive and neutrally charged lipophilic (tending to combine with lipids, oils, or fats) compounds. Testosterone is very lipophilic, especially before the cypionate ester is cleaved. Some of your free testosterone may actually be bound by CBG and orosomucoid, but this is ignored by the medical community. The characteristics of testosterone binding to CBG and orosomucoid and the biological roles of these binding proteins in regulating testosterone bioavailability remain incompletely understood.
There are two variants of albumin; Albumin Catania and albumin Roma. Albumin Roma has a low binding affinity to testosterone, but it is unknown if albumin Catania has an increased or decreased affinity. The free hormone hypothesis postulates that there is a 1:1 binding stoichiometry of testosterone to albumin, or that one testosterone molecule binds to one albumin. Multiple domains of albumin are involved in the binding and transport of biomolecules; albumin has three binding pockets. More recently, concluded on the basis of studies, the data indicated the existence of cooperativity between secondary binding sites and the primary testosterone binding site. For many ligands (a molecule that binds to a receptor), the multiple binding sites are allosterically coupled, or the binding of ligands to multiple sites changes the binding affinity and dissociation capability. It is conceivable that testosterone, like other ligands, may also have multiple binding sites with distinct affinities on albumin. Oversimplification of binding models and potentially erroneous assumptions can have major implications not only on estimates of testosterone’s bioavailability but also on putative competitive interactions with fatty acids and other hormones and drugs.
The free hormone hypothesis outlines a simplistic role for SHBG, hypothesizing that one testosterone molecule binds to one SHBG protein and is subsequently nullified. Speculation of what happens to testosterone bound SHBG is absent; the presumption being that there is no further effect.
SHBG is universally known as a transporter protein. Testosterone is produced in the testicles in males, and in the ovaries and adrenal glands in females. Without transporter proteins, testosterone would have no effect on the androgen receptors of cells throughout the body, including in the brain. If SHBG did not transport testosterone from one place to another, if it was permanently inactivated testosterone, then it would be a suicide protein, or an antagonist protein. Testosterone may dissociate from SHBG so it can be utilized by cells, making the total testosterone concentration bound by SHBG a more important metric than is assumed.
Studies have demonstrated that SHBG is a homodimeric protein, meaning it consists of two identical protein subunits. Each subunit has a testosterone binding domain, indicating that SHBG binds two testosterone molecules. Additionally, these two ligands binding to the SHBG dimer involves a complex allosteric mechanism. This means that binding two testosterone molecules to SHBG causes a conformational change that alters the binding affinity of testosterone. Recent evidence derived from new biophysical techniques indicates that the binding of testosterone to SHBG is a dynamic, multistep process. The binding of one molecule of testosterone to the first binding site on an SHBG dimer leads to conformational rearrangement and allostery between the two binding sites, such that the second testosterone molecule binds to the second binding site with a different binding affinity. Testosterone’s bond to SHBG may not be as strong as previously thought. Moreover, Complimentary studies show that the binding affinity of testosterone to SHBG may change depending on total SHBG concentration, acidity, and temperature.
Most importantly, evidence suggests that testosterone bound SHBG is utilized by cells potentially through three separate mechanisms: 1) SHBG-bound testosterone is internalized into the cell through an endocytic process mediated by the membrane protein megalin. Testosterone bound SHBG binds to megalin on the cell surface, is internalized, absorbed by a lysosome, and testosterone is released from SHBG at the low pH within the lysosome, which then binds to the androgen receptor; 2) SHBG potentially binds to a receptor at the cell surface, which then binds testosterone, and a G-coupled protein activates cAMP and PKA. PKA can modulate androgen receptor functions through phosphorylation, which increases the frequency of available androgen receptors and increases the binding affinity of testosterone to the AR. Both of these functions increase androgen sensitivity; 3) Testosterone bound SHBG may bind to the cell matrix associated proteins fibulin and integrin. This binding signals the cell to regulate cell adhesion, cell proliferation, and cell migration, which would control steroid access to target cells and regulate bioavailability of testosterone to these cells – which increases androgen sensitivity and testosterone’s effectiveness.
In summary, evidence suggests that testosterone bound SHBG is utilized by cells through multiple mechanisms and is necessary for optimal function. Because of this, total testosterone is a more important metric to consider than free testosterone when considering testosterone therapy. If total testosterone is 600 or 700ng/dL, and SHBG is elevated, then free testosterone will be low, and there will be symptoms of hypogonadism. Free testosterone is a metric that should be considered, but testosterone bound SHBG needs to be considered as well. Too much free testosterone without the presence of SHBG will downregulate gene transcription for androgen receptors, reducing the frequency of androgen receptors, thereby reducing androgen sensitivity. If total testosterone is in the 400’s or 500’s, and SHBG on the lower end, making free testosterone in the middle to the high end of the range, then symptoms of hypogonadism may be present because there is not enough testosterone bound SHBG in circulation. This is the context in which the importance of total testosterone has been misclassified and hypogonadism historically has been misdiagnosed because of the free hormone hypothesis.
Equilibrium dialysis is widely considered the gold standard method of directly testing free testosterone in the blood. Equilibrium dialysis involves dialysis of a serum sample across a semipermeable membrane where protein bound testosterone is retained, and free testosterone permeates the membrane. The free testosterone can then be measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. However, this test is not widely available, it is technically demanding, and its performance is affected by assay conditions which can result in high assay variability. In other words, equilibrium dialysis, the best direct test for free testosterone, is not widely available, is expensive, and is very susceptible to errors which lead to inaccuracy. Every other direct blood test is less accurate than equilibrium dialysis.
Most hospital and commercial laboratories do not offer an equilibrium dialysis assay for free testosterone, most likely because of operational complexities in performing the assay and difficulties in automating the procedure. Recognizing the practical difficulties that practicing clinicians face in obtaining precise and accurate measurements of free testosterone concentrations by the equilibrium dialysis method, an expert panel of the Endocrine Society concluded that “calculated free testosterone, using high quality testosterone and SHBG assays with well-defined reference intervals, is the most useful clinical marker…”. However, the current algorithms used in calculated free testosterone assume a linear binding model, that testosterone binds to cognate proteins with a constant binding affinity, and the calculations assume a 1:1 stoichiometry, or that one testosterone molecule binds to one SHBG and one albumin. As stated earlier, SHBG and albumin have multiple binding sites and each site binds with substantially different affinities. If SHBG and albumin have multiple binding sites each with different binding affinities, but calculated free testosterone assumes a linear binding model in which one testosterone molecule binds to one SHBG and one albumin, then the calculation of free testosterone may be inaccurate by more than 50%.
All methods of measuring free testosterone are riddled with potential inaccuracies making free testosterone an unreliable and untrustworthy metric.
Sex steroid bioactivity and the respective roles of SHBG and albumin are more complex than originally believed. The oversimplified assumptions of stoichiometry, binding dynamics, and binding affinity have contributed to the development of inaccurate linear binding models, which have been propagated without much critical reappraisal until now. These historical linear models and the resulting equations for calculating free testosterone based on these legacy models are widely used and have increased the risk of misclassifying those seeking testosterone therapy. Consequently, symptoms of hypogonadism and low testosterone, in men and women, have been misdiagnosed, undiagnosed, and mistreated for many decades.