Measurement of free thyroxine in human serum by a Sephadex binding method.

Abstract

ABSTRACT A method of measuring free thyroxine (T4) is described which uses the property of Sephadex to bind T4. If 500 mg Sephadex G-25, 40 μI of serum in 4 ml physiological buffer, and 125I-T4 are mixed for 10 min at 37 C in a test tube, the unbound or free 125I-T4 in the aqueous phase is 12% of the 125I-T4 bound to Sephadex. This fraction depends specifically on the low affinity but high capacity of Sephadex for T4, and is unaffected by the amount of T4 or protein in the system although the amount of protein-bound 125I-T4 in the aqueous phase may change markedly. Since Sephadex-bound 125I-T4 is readily measured, the amount of free 125I-T4, and the fraction, free/total 125I-T4 in the dilute serum which forms the aqueous phase in the system are readily calculated. This “free T4 fraction” is related to the in vivo free T4 fraction only if the ratio, total T4: T4 binding protein is the same in this aqueous phase as it is in undiluted serum. These conditions are obtainable by experimental design or calculati...