Influence of adsorption and deproteination on potential free thyroxine reference methods.
BACKGROUND
There is a need for consensus concerning reference methods to be used for calibration of commercial free-thyroxine (FT(4)) assays.


METHODS
Three different potential reference techniques were investigated for adsorption of T(4) to membrane materials, including any in vitro solid surfaces to which T(4) might adsorb, and for efficient separation of the T(4)-binding proteins from the free hormone fraction. We compared ultrafiltration with different commercial ultrafiltration units, ultrafiltration by dialysis tubing, equilibrium dialysis, and ultracentrifugation. We measured the adsorption to membranes and materials with L-[(125)I]T(4). Separation efficiency was determined by measuring the T(4)-binding protein albumin in the ultrafiltrate and the dialysate as well as in the supernatant from the ultracentrifugation with a double-antibody sandwich ELISA technique.


RESULTS
We found a constant relationship between the amount of T(4) adsorbed to the dialysis or ultrafiltration membranes/materials and the initial T(4) concentration in HEPES buffer (protein-free medium). T(4) was considerably less adsorbed from serum than from HEPES buffer (P <0.001). Serum T(4) was less adsorbed upon ultracentrifugation than during dialysis and ultrafiltration (P <0.001). It was difficult to completely separate FT(4) from the binding proteins by ultrafiltration and ultracentrifugation. Separation by ultrafiltration depended on the material used.


CONCLUSIONS
No investigated separation technique provides technically and theoretically correct separation of the free fraction of T(4) from the protein-bound fraction. Equilibrium dialysis seems to be the least compromised.
Holm
Steen S
SS
Department of Clinical Biochemistry, Holbaek Hospital, 4300 Holbaek, Denmark. chstho@vestamt.dk
Andreasen
Lisbeth
L
Hansen
Steen H
SH
Faber
Jens
J
Staun-Olsen
Poul
P
D000327:Adsorption; D000418:Albumins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003956:Dialysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008567:Membranes, Artificial; D012015:Reference Standards; D013963:Thyroid Hormones; D013974:Thyroxine / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism; D014462:Ultrafiltration; D000097810:Thyroid Hormone-Binding Proteins
D000418:Albumins; D002352:Carrier Proteins; D008565:Membrane Proteins; D008567:Membranes, Artificial; D013963:Thyroid Hormones; D013974:Thyroxine; D000097810:Thyroid Hormone-Binding Proteins