Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis.
Level 3 - non-randomized controlled study
Cross-sectional analytical validation study comparing calculation models to direct reference laboratory measurements.
PubMed 29618085 · doi:10.1210/jc.2017-02360
What was done
Free testosterone (FT) was directly measured in serum samples from 183 women and 146 men using equilibrium dialysis (ED) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Direct ED-FT values were compared against three free testosterone calculation models: the Vermeulen law of mass action equation (cFT-V), the Ly empirical equation (cFT-L), and the Zakharov multistep dynamic allosteric model (cFT-Z).
What was found
Direct ED-FT median concentrations were 13 pmol/L (1.2% of total testosterone) in women and 248 pmol/L (1.5% of total testosterone) in men. In male samples, the median (2.5th to 97.5th percentile) ratios of calculated FT over direct ED-FT were 1.19 (0.9 to 1.47) for cFT-V, 1.00 (0.69 to 1.42) for cFT-L, and 2.05 (1.26 to 3.26) for cFT-Z. The ratio for cFT-V was not significantly affected by SHBG, total testosterone, or albumin concentrations (ρ range, -0.01 to 0.17). Ratios for cFT-L and cFT-Z were significantly affected (P < 0.05 and P < 0.001, respectively) and strongly correlated with SHBG levels (ρ = 0.72 and 0.75, respectively). Rank correlation between calculated FT% and ED-FT% in men was 0.89 for cFT-V, 0.74 for cFT-L, and 0.62 for cFT-Z.
Why it matters
Vermeulen's equation overestimates equilibrium dialysis free testosterone by roughly 19% but provides the most robust calculation across varying SHBG, albumin, and total testosterone levels. Conversely, the newer Zakharov allosteric model substantially overestimates free testosterone twofold and introduces strong SHBG-dependent bias.
Limits
The study is limited to 329 serum samples, and the abstract only presents calculation-ratio performance data for men, omitting numerical ratio results for women. Clinical outcomes, diagnostic cutoffs for endocrine disorders, and inter-laboratory analytical variability were not evaluated.
Cited by
- supports Equilibrium dialysis and equilibrium ultrafiltration are direct measurement assays for free testosterone rather than calculated estimates.