Reassessing Free-Testosterone Calculation by Liquid Chromatography–Tandem Mass Spectrometry Direct Equilibrium Dialysis
Abstract
Context Assessment of free testosterone (FT) might help evaluate androgen status in patients with borderline total testosterone (T) and/or altered sex hormone-binding globulin (SHBG) levels. However, the validity of different methods to measure FT is debatable. Methods Serum from 183 women and 146 men was analyzed using equilibrium dialysis (ED), with FT directly measured by liquid chromatography-tandem mass spectrometry. FT calculation was re-evaluated for the mass action law-based equation according to Vermeulen (cFT-V), empirical equations according to Ly (cFT-L), and a proposed calculation based on a multistep, dynamic, allosteric model according to Zakharov (cFT-Z). Results FT level analyzed by ED [median,13 pmol/L (1.2% of T) in women; 248 pmol/L (1.5% of T) in men] was strongly inversely correlated to SHBG level, significantly to albumin level in women, and only weakly to SHBG level in men. The median [percentile (p) range, 2.5 to 97.5] ratios of calculated FT (cFT) over ED-FT (from European Male Aging Study samples) were 1.19 (0.9 to 1.47), 1.00 (0.69 to 1.42), and 2.05 (1.26 to 3.26) for cFT-V, cFT-L, and cFT-Z, respectively. The ratio for cFT-V was not significantly affected by SHBG, T, or albumin levels (ρ range, 0.17 to -0.01); ratios for cFT-L and cFT-Z were affected (P Conclusion FT results by direct ED confirm prior FT data from indirect ED and ultrafiltration methodologies. Calculations have inherent limitations, with clinically important differences among eva…
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The Ly/Handelsman and Zakharov methods do not operate independent of SHBG, total T, and to a lesser extent albumin values.
“Our data also demonstrated that both Ly/Handelsman and Zakharov methods do not operate independent of SHBG, total T and to a lesser extent albumin values [221].”
From Role of sex hormone-binding globulin in the free hormone hypothesis and the relevance of free testosterone in androgen physiology (2022) · cites this paper as [221]
The Vermeulen formula overestimates free T by 20-30% but is independent of SHBG, total T, and albumin.
“In contrast we concluded that, although overestimating free T by 20–30%, the formula according to Vermeulen was independent of SHBG, total T and albumin [221].”
From Role of sex hormone-binding globulin in the free hormone hypothesis and the relevance of free testosterone in androgen physiology (2022) · cites this paper as [221]
In a Belgian study of over 300 serum samples, median bias (ratio of calculated to measured free T) was 1.19 for Vermeulen, 1.0 for Ly/Handelsman, and 2.05 for Zakharov.
“Median bias, expressed by the ratio of calculated to measured free T, was 1.19, 1.0 and 2.05 for the Vermeulen, Ly/Handelsman and Zakharov model, respectively.”
From Role of sex hormone-binding globulin in the free hormone hypothesis and the relevance of free testosterone in androgen physiology (2022) · cites this paper as [221]
There is fairly good agreement between measured free T and free T calculated by the Vermeulen formula when validated against ED.
“There is, however, a fairly good agreement between measured and free T calculated by the Vermeulen formula, if validated against ED [221, 228].”
From Role of sex hormone-binding globulin in the free hormone hypothesis and the relevance of free testosterone in androgen physiology (2022) · cites this paper as [221]
Total testosterone, SHBG, free androgen index, calculated free testosterone, directly measured free testosterone, and bioactive or bioavailable testosterone do not represent interchangeable endpoints.
“Consequently, TT, SHBG, free androgen index (FAI), calculated free testosterone (calculated FT), directly measured FT, and bioactive or bioavailable testosterone do not represent interchangeable endpoints.”
Several androgen outcomes were secondary biomarkers.
“These issues are particularly relevant because several androgen outcomes were secondary biomarkers, and because 25(OH)D and testosterone assays varied across trials [3,10,35,36].”
25(OH)D and testosterone assays varied across trials.
“These issues are particularly relevant because several androgen outcomes were secondary biomarkers, and because 25(OH)D and testosterone assays varied across trials [3,10,35,36].”
Directly measured free testosterone and calculated free testosterone are analytically non-equivalent and may differ in calibration, assay performance, assumptions, and dependence on total testosterone, SHBG, and albumin.
“Directly measured FT and calculated FT were not pooled because they are analytically non-equivalent and may differ in calibration, assay performance, assumptions, and dependence on TT, SHBG, and albumin [8,9,10].”
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- Authors
- Fiers, T., Wu, F., Moghetti, P., Vanderschueren, D., Lapauw, B., & Kaufman, J.-M.
- Affiliations
- Ghent University Hospital (BE)
- Manchester Academic Health Science Centre (GB)
- University of Verona (IT)
- KU Leuven (BE)
- Venue
- The Journal of Clinical Endocrinology & Metabolism
- Publisher
- The Endocrine Society
- Published
- 2018-03-29
- Volume
- 103
- Issue
- 6
- Pages
- 2167-2174
- ISSN
- 0021-972X (ISSN-L)
- 1945-7197
- OpenAlex
- W2795170679
- Cited by
- 70
- References
- 10
- Citations by year
- 2026: 8 · 2025: 15 · 2024: 14 · 2023: 1 · 2022: 14 · 2021: 7 · 2020: 4 · 2019: 4 · 2018: 2
- FWCI
- 3.38
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Clinical Endocrinology & Metabolism (publishedVersion)
- PubMed (publishedVersion)
- Ghent University Academic Bibliography (Ghent University) (submittedVersion)
- Lirias (submittedVersion)
- Research Explorer (The University of Manchester) (submittedVersion)
- Topics
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Keywords
- free testosterone, equilibrium dialysis, sex hormone-binding globulin, total testosterone, albumin binding, mass action law, androgen status, method comparison, ultrafiltration, liquid chromatography-tandem mass spectrometry
- MeSH terms
- Adolescent, Adult, Aged, Aging, Chromatography, Liquid, Female, Humans, Male, Middle Aged, Polycystic Ovary Syndrome, Sex Hormone-Binding Globulin, Testosterone, Tandem Mass Spectrometry, Young Adult
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