A Critical Evaluation of Simple Methods for the Estimation of Free Testosterone in Serum

Vermeulen, Alex; Verdonck, Lieve; Kaufman, Jean M.

doi:10.1210/jcem.84.10.6079

Cited by 3,824Hormonal and reproductive studiesAdrenal Hormones and DisordersClinical Laboratory Practices and Quality Control

A Critical Evaluation of Simple Methods for the Estimation of Free Testosterone in Serum

Vermeulen, Alex, Verdonck, Lieve, Kaufman, Jean M.·1999·The Journal of Clinical Endocrinology & Metabolism

DOI 10.1210/jcem.84.10.6079

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Abstract

The free and nonspecifically bound plasma hormone levels generally reflect the clinical situation more accurately than total plasma hormone levels. Hence, it is important to have reliable indexes of these fractions. The apparent free testosterone (T) concentration obtained by equilibrium dialysis (AFTC) as well as the fraction of serum T not precipitated by 50% ammonium sulfate concentration (non-SHBG-T; SHBG, sex hormone-binding globulin), often referred to as bioavailable T, appear to represent reliable indexes of biologically readily available T, but are not well suited for clinical routine, being too time consuming. Several other parameters have been used without complete validation, however: direct immunoassay of free T with a labeled T analog (aFT), calculation of free T (FT) from total T and immunoassayed SHBG concentrations (iSHBG), and the free androgen index (FAI = the ratio 100T/iSHBG). In the view of substantial discrepancies in the literature concerning the free or bioavailable T levels, we compared AFTC, FT, aFT, FAI, and non-SHBG-T levels in a large number of sera with SHBG capacities varying from low, as in hirsute women, to extremely high as in hyperthyroidism. All these indexes of bioavailable T correlated significantly with the AFTC concentration; AFTC and FT values were almost identical under all conditions studied, except during pregnancy. Values for aFT, however, were only a fraction of either AFTC or FT, the fraction varying as a function of SHBG levels…

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Free T algorithms can be divided into three categories: linear mass action, non-linear allosteric, and empirical bootstrap.
“A wide spectrum of algorithms for free T exists, which can broadly be divided into three categories (Table 6).”
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 [208]
Variability and assumptions in binding affinity, stoichiometry, and allosteric interaction produce different quantities of free T in different models.
“Second, concerning the interaction between SHBG and T, variability and assumptions in terms of binding affinity, stoichiometry and allosteric interaction produce different quantities of free T in different models [5, 208].”
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 [208]
Because bioavailable T measurement is not standardized, large variability in bioavailable T results may exist between laboratories.
“Furthermore, because it is not standardized, large variability in bioavailable T results may exist between laboratories [208].”
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 [208]
The first category of free T equations was derived from the general law of mass action, with association constants for albumin and SHBG obtained from in vitro experiments by Scatchard analysis.
“First, several equations were derived from the general law of mass action. Association constants for albumin and SHBG used in these formulas were obtained from in vitro experiments by linearizing data through Scatchard analysis [16, 208, 212].”
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 [208]
Analog-based immunoassays do not detect nor quantify free T.
“Comparative studies of this analog-based immunoassay and ED confirmed that this technique does not detect nor quantify free T and that is should, therefore, not be used as an assay for free T [205, 207, 208].”
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 [208]

Free testosterone values were calculated from total testosterone, SHBG, and albumin according to Vermeulen.
“FT values were calculated from TT (measured by ID-LC–MS/MS), SHBG, and albumin according to Vermeulen [20].”
From Effects of vitamin D supplementation on androgens in men with low testosterone levels: a randomized controlled trial (2019) · cites this paper as [20]
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.”
From Vitamin D Supplementation, Total Testosterone, and Androgen Bioavailability Markers in Adult Men: A Systematic Review and Meta-Analysis of Randomized Controlled Trials (2026) · cites this paper as [8]
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].”
From Vitamin D Supplementation, Total Testosterone, and Androgen Bioavailability Markers in Adult Men: A Systematic Review and Meta-Analysis of Randomized Controlled Trials (2026) · cites this paper as [8]
Calculated free testosterone values are a reliable index of free testosterone.
“This calculation was recently evaluated by different investigators, and found to be a reliable index of free testosterone (Vermeulen et al., 1999;Morley et al., 2002).”
From Endogenous testosterone levels and smoking in men. The fifth Tromsø study (2007) · cites this paper as Vermeulen et al., 1999
The analyses were adjusted for cigarette smoking and time of abstinence for semen parameters, and for smoking and cohort for hormones.
“We used general linear models, with adjustments including cigarette smoking, and for semen parameters also abstinence time.”
From Decreased sperm counts in Swedish users of oral tobacco (2022) · cites this paper as [28]
Endurance athletes may have lower concentrations of free and total testosterone than non-athletes.
“Additionally, endurance athletes may have lower concentrations of free and total testosterone than non-athletes [24, 25].”
From Association of cigarette smoking, alcohol consumption, and physical activity with sex steroid hormone levels in US men (2009) · cites this paper as [25]
Free testosterone calculated using the formula of Vermeulen and colleagues is a useful tool for assessment of biochemical hyperandrogenism when direct free testosterone assays are not entirely reliable.
“As direct assays to measure free testosterone are not entirely reliable, another useful tool for the assessment of biochemical hyperandrogenism is free testosterone calculated using the formula of Vermeulen and collogues [36].”
From Controversies in the Pathogenesis, Diagnosis and Treatment of PCOS: Focus on Insulin Resistance, Inflammation, and Hyperandrogenism (2022) · cites this paper as [36]
A major challenge in testosterone interpretation is significant variability among laboratories and assay techniques.
“One of the challenges with T interpretation is the significant variability existing among laboratories and various assay techniques.”
From Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials (2016) · cites this paper as [88]
At lower concentrations, immunoassays show particularly significant variation among techniques, with equivalent samples producing 2.7-14.3 fold variations in reported results.
“At lower concentrations, IA demonstrate particularly significant variations among techniques, with equivalent samples resulting in 2.7–14.3 fold variations in reported results.”
From Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials (2016) · cites this paper as [88]
Calculated free testosterone levels correlate well with measurements of free testosterone as determined by equilibrium dialysis.
“The cFT levels (20) correlate well with measurements of free testosterone as determined by the gold standard of equilibrium dialysis (22).”
From Low testosterone levels are common and associated with insulin resistance in men with diabetes (2008) · cites this paper as [20]

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A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab
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Discrepancies in Four Algorithms for the Calculation of Free and Bioavailable Testosterone
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Authors: Vermeulen, A., Verdonck, L., & Kaufman, J. M.
Affiliations: Ghent University Hospital (BE)
Corresponding author: Kaufman, Jean M.
ORCID: Vermeulen, Alex
Kaufman, Jean M.
Venue: The Journal of Clinical Endocrinology & Metabolism
Publisher: The Endocrine Society
Published: 1999-10-01
Volume: 84
Issue: 10
Pages: 3666-3672
ISSN: 0021-972X (ISSN-L)
1945-7197
DOI: 10.1210/jcem.84.10.6079
OpenAlex: W2098247759
Cited by: 3,824
References: 15
Citations by year: 2026: 107 · 2025: 131 · 2024: 122 · 2023: 137 · 2022: 161 · 2021: 161 · 2020: 170 · 2019: 141 · 2018: 144 · 2017: 165 · 2016: 168 · 2015: 196 · 2014: 212 · 2013: 206 · 2012: 204
FWCI: 22.21
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)
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, bioavailable testosterone, sex hormone-binding globulin, equilibrium dialysis, free androgen index, ammonium sulfate precipitation, pregnancy, hyperthyroidism, hirsutism
MeSH terms: Adult, Aged, Aged, 80 and over, Androgens, Dialysis, Evaluation Studies as Topic, Female, Humans, Hyperthyroidism, Immunoassay, Male, Methods, Middle Aged, Osmolar Concentration, Pregnancy, Sex Hormone-Binding Globulin, Testosterone, Postmenopause

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A Critical Evaluation of Simple Methods for the Estimation of Free Testosterone in Serum

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