madman
Super Moderator
* In this analysis, we describe the clinical and biochemical features of men with markedly elevated SHBG levels who exhibited unequivocally low free testosterone concentrations, measured using rigorously validated equilibrium dialysis assay. Despite unequivocally low free testosterone concentrations, some of these men showed mid- to high-normal or even total testosterone levels above the reference range, creating a striking divergence between total and free testosterone concentrations. Notably, all met predefined criteria for primary hypogonadism (1). These findings are consistent with free testosterone—rather than total testosterone—serving as the physiologically relevant regulator of the hypothalamic-pituitary-testicular feedback loop.
* A key barrier to wider free testosterone adoption is the limited access to accurate equilibrium dialysis method, compounded by inter-laboratory variability and variable non-harmonized reference ranges reported by commercial and academic laboratories. Although algorithms for estimating free testosterone concentrations from total testosterone, SHBG, and albumin concentrations have been published (30-33), such equations have inherent limitations because of the dynamic changes in the apparent binding affinity of SHBG and human serum albumin for sex hormones with varying sex hormone and binding protein concentrations (32,34-36). The findings of our analyses provide a strong rationale for the generation of harmonized reference ranges for free testosterone concentration using standardized equilibrium dialysis procedure coupled with the measurements of testosterone in the dialysate using an LC-MS/MS assay certified by the HoST Program, such as that which we have previously published (11).
INTRODUCTION
Testosterone deficiency manifests as a clinical syndrome in men due to dysfunction at any level of the hypothalamic-pituitary-testicular (HPT) axis, leading to inadequate testicular secretion of testosterone (1). Therefore, accurate measurement of testosterone concentrations is essential for the diagnosis of testosterone deficiency. Circulating testosterone exists predominantly bound to sex hormone-binding globulin (SHBG) and albumin, with lesser fractions bound to orosomucoid and corticosteroid-binding globulin, and only 1-4% circulating as unbound free testosterone (2). The Free Hormone Hypothesis (FHH) posits that the free testosterone is able to rapidly diffuse across the plasma membrane to activate intracellular signaling, producing tissue-specific effects, and is, therefore, the biologically active fraction of circulating testosterone (3,4).
An important corollary of the FHH is that free rather than total testosterone serves as the key mediator, directly as well as through its conversion to estradiol, of the negative feedback on the HPT axis through central biosensors in hypothalamic KNDy neurons and pituitary gonadotropes. Consequently, when free testosterone concentration is within the physiological range—irrespective of SHBG or total testosterone concentrations—men should exhibit clinical eugonadism with normal luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations; conversely, subnormal free testosterone should drive compensatory elevations in LH and/or FSH, independent of total testosterone or SHBG.
An additional corollary of the free hormone hypothesis is that free testosterone concentration should outperform total testosterone concentration as a circulating biomarker of systemic androgen action. However, total and free testosterone concentrations display strong collinearity (r = 0.7–0.96 in healthy young men across assays), while associations between testosterone changes and testosterone-responsive endpoints such as sexual desire and sexual activity remain modest (r = 0.1–0.3) (5-8). For example, in the Testosterone Trials (TTrials) (6,7), the increments in free testosterone concentrations above baseline correlated modestly more robustly with sexual activity improvements than changes in total testosterone, though both showed positive, SHBG-independent association—highlighting how collinearity between total and free testosterone confounds these correlational analyses in observational and interventional studies and fuels debates over the superiority of free testosterone over total testosterone (9).
Conclusions
In men with markedly elevated SHBG, low free testosterone concentrations were consistently associated with one or more symptoms and signs of testosterone deficiency and with elevated LH and/or FSH concentrations, even when total testosterone concentrations were within or above the reference range. Taken together, these data support the primacy of circulating free testosterone over total testosterone as the biologically relevant determinant of the systemic manifestations of testosterone deficiency, including the feedback regulation of gonadotropin secretion. Our findings provide a strong rationale for expanding access to accurate, validated free testosterone assays with rigorously derived, harmonized reference ranges to improve diagnostic precision and patient care.
* A key barrier to wider free testosterone adoption is the limited access to accurate equilibrium dialysis method, compounded by inter-laboratory variability and variable non-harmonized reference ranges reported by commercial and academic laboratories. Although algorithms for estimating free testosterone concentrations from total testosterone, SHBG, and albumin concentrations have been published (30-33), such equations have inherent limitations because of the dynamic changes in the apparent binding affinity of SHBG and human serum albumin for sex hormones with varying sex hormone and binding protein concentrations (32,34-36). The findings of our analyses provide a strong rationale for the generation of harmonized reference ranges for free testosterone concentration using standardized equilibrium dialysis procedure coupled with the measurements of testosterone in the dialysate using an LC-MS/MS assay certified by the HoST Program, such as that which we have previously published (11).
INTRODUCTION
Testosterone deficiency manifests as a clinical syndrome in men due to dysfunction at any level of the hypothalamic-pituitary-testicular (HPT) axis, leading to inadequate testicular secretion of testosterone (1). Therefore, accurate measurement of testosterone concentrations is essential for the diagnosis of testosterone deficiency. Circulating testosterone exists predominantly bound to sex hormone-binding globulin (SHBG) and albumin, with lesser fractions bound to orosomucoid and corticosteroid-binding globulin, and only 1-4% circulating as unbound free testosterone (2). The Free Hormone Hypothesis (FHH) posits that the free testosterone is able to rapidly diffuse across the plasma membrane to activate intracellular signaling, producing tissue-specific effects, and is, therefore, the biologically active fraction of circulating testosterone (3,4).
An important corollary of the FHH is that free rather than total testosterone serves as the key mediator, directly as well as through its conversion to estradiol, of the negative feedback on the HPT axis through central biosensors in hypothalamic KNDy neurons and pituitary gonadotropes. Consequently, when free testosterone concentration is within the physiological range—irrespective of SHBG or total testosterone concentrations—men should exhibit clinical eugonadism with normal luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations; conversely, subnormal free testosterone should drive compensatory elevations in LH and/or FSH, independent of total testosterone or SHBG.
An additional corollary of the free hormone hypothesis is that free testosterone concentration should outperform total testosterone concentration as a circulating biomarker of systemic androgen action. However, total and free testosterone concentrations display strong collinearity (r = 0.7–0.96 in healthy young men across assays), while associations between testosterone changes and testosterone-responsive endpoints such as sexual desire and sexual activity remain modest (r = 0.1–0.3) (5-8). For example, in the Testosterone Trials (TTrials) (6,7), the increments in free testosterone concentrations above baseline correlated modestly more robustly with sexual activity improvements than changes in total testosterone, though both showed positive, SHBG-independent association—highlighting how collinearity between total and free testosterone confounds these correlational analyses in observational and interventional studies and fuels debates over the superiority of free testosterone over total testosterone (9).
Conclusions
In men with markedly elevated SHBG, low free testosterone concentrations were consistently associated with one or more symptoms and signs of testosterone deficiency and with elevated LH and/or FSH concentrations, even when total testosterone concentrations were within or above the reference range. Taken together, these data support the primacy of circulating free testosterone over total testosterone as the biologically relevant determinant of the systemic manifestations of testosterone deficiency, including the feedback regulation of gonadotropin secretion. Our findings provide a strong rationale for expanding access to accurate, validated free testosterone assays with rigorously derived, harmonized reference ranges to improve diagnostic precision and patient care.
