Background: Direct free thyroxine (T4) measurements have been linked to both T4-binding serum protein concentrations and protein-bound T4 concentrations. Whether this is evidence of a relationship to total T4 concentrations has not been reported. Methods: We compared an analog-based direct free T4 immunoassay and a total T4 immunoassay. Each assay was applied to the fractions of serum T4 obtained by ultrafiltration and equilibrium dialysis. Both were applied to serum-based solutions in which free T4, T4-binding proteins, protein-bound T4, and total T4 were systematically varied, held constant, or excluded. Results: Neither the free T4 assay nor the total T4 assay detected dialyzable or ultrafilterable serum T4. Both assays detected and reported the T4 retained with serum proteins. Both free and total T4 results were related to the same total T4 concentrations in the presence and absence of T4-binding proteins. Both results were similarly related to total T4 concentrations when free T4 was held constant while total T4 was varied. Both were similarly related to a total T4 concentration that was held constant while free T4 progressively replaced protein-bound T4. These free T4 results, like total T4 results, were unresponsive to a 500-fold variation in dialyzable T4 concentrations. Conclusion: New experiments extend the characterization of a longstanding and incompletely characterized analog-based free T4 immunoassay. These free T4 measurements relate to total T4 concentrations in the same way that total T4 measurements do.

Abstract Background: Direct equilibrium dialysis and direct ultrafiltration free thyroxine (T4) assays rely on semipermeable membranes to exclude T4-binding serum proteins from dialysates and ultrafiltrates. The presence of these proteins in dialysates or ultrafiltrates will yield spuriously high free T4 values when free T4 is quantified by RIA. Methods: We used a nonanalog free T4 RIA that detects and quantifies dialyzable and ultrafilterable serum free T4 to detect T4-binding serum proteins. Two equilibrium dialysis devices and 3 ultrafiltration devices were used to illustrate this application. Displacements of [125I]T4 from anti-T4 by various concentrations of T4-depleted thyroxine-binding globulin, albumin, and serum total protein were compared to displacements by various concentrations of free T4. Results: Both dialysis devices excluded detectable T4-binding serum proteins from dialysates. Two of 3 ultrafiltration devices excluded detectable T4-binding serum proteins from ultrafiltrates. One did not, and its ultrafiltrate yielded spurious free T4 values that correlated directly with serum protein concentrations. Conclusion: The presence or absence of T4-binding proteins in dialysates and ultrafiltrates and the spurious free T4 values that these proteins cause can be documented using a nonanalog free T4 RIA.

The first evidence of unwanted serum protein effects on analog-based total T4 determinations came from a study that varied serum protein concentrations while total T4 concentrations were constant (4). The present study approached this issue by varying total T4 concentrations while protein concentrations were constant. Four analog-based total T4 immunoassays were applied to solutions that contained either free T4 without binding protein, a T4 binding protein without T4, or protein bound T4. When total T4 concentrations were 3-12 µg/dL, the assays reported total T4 determinations that ranged from none detected to 23 µg/dL. These T4 determinations reflected the protein to which T4 was bound, in addition to the level of T4. Total T4 was under-represented when T4 was unbound, or TBG bound. Total T4 was over-represented when T4 was albumin bound, or transthyretin bound. There were substantial disparities among assays applied to the same total T4 solutions. These assays reported no detectable T4 when applied to T4 binding protein solutions without protein bound T4. Nonetheless, T4 binding proteins contributed to the underestimates and overestimates of protein bound T4. Different forms of protein bound T4 were quantified differently, evidence that protein-T4 complexes persist during quantification. We attribute the unexpected total T4 values to a combination of incomplete protein bound T4 release from T4 binding proteins during quantification, and variably inaccurate quantifications of the protein bound T4 that remained.

Clinical laboratories often use analog-based immunoassays to estimate serum free T4 concentrations. These assays yield free T4 estimates that correlate closely with T4 binding protein concentrations (1-11). This correlation implies that either T4 binding proteins or protein bound T4 contribute to analog-based free T4 values. To further study the contributions made by T4 binding proteins to these free T4 estimates, four analog-based free T4 assays were applied to: 1) free T4 solutions without T4 binding proteins, 2) to T4 binding protein solutions without T4, and 3) to total T4 solutions containing T4 binding protein, free T4, and protein bound T4. The free T4 estimates obtained with these solutions ranged from 0.2-8.6 ng/dL, when free T4 concentrations ranged from