The metabolism of L-thyroxine (T4) and L-triiodothyronine (T3) by human fibroblasts in tissue culture was studied. Fibroblasts were incubated in serumless medium and in media containing various concentrations of normal human serum (HS) or fetal calf serum (FCS) enriched with both T4-125I and T3-131I, and the simultaneous cell uptake of both tracers and the T4 to T3 conversion determined. Interference from the preferential cellular uptake of T3 impurities in T4 was corrected by determination of the 125I/131I ratio migrating as T3 on bidimensional paper chromatography of medium and cells. In the absence of serum in the medium, cell to medium (C/M) concentration gradient was 90 to 100 for T3 and 49 to 58 for T4. With increasing amounts of HS, the fractional uptake of T3 and T4 diminished and the C/M gradient for T4 was less than 1. The cellular T3/T4 ratio increased to a maximum of 11.4 in the presence of 5% HS. The T3/T4 ratio in the dialysable fraction was 1.01 for serumless medium and 5.8 for medium containing 10% HS. Data indicate that cellular binding sites have higher affinity for T3 than T4; an observation supported by the electrophoretic identification of distinct cellular T3 binding protein in sonicated fibroblasts. In all experiments the 125I/131I ratio in the T3 zone was higher in cells than in medium. The corrected “apparent” T4 to T3 conversion was from 0.4 to 5.7%. Addition of up to 500 fold increments of T4 to the medium did not result in saturation of the cellular monodeiodination capacity. Also nonphysiological concentrations of T3 had no apparent inhibitory effect on the T4 to T3 conversion. “Chase”-labeling experiments revealed a rapid equilibration of the isotopes, accompanied by a relative increase in the proportion of T3 retained. These studies demonstrate the effect of cellular binding proteins with higher affinity for T3 than T4. They also emphasize two problems in the estimation of the net T4 to T3 conversion: (a) the observed percent conversion may be over estimated if not corrected for the preferential cellular uptake of T3 contaminant or (b) under estimated, as in the present study, when the rate of cellular T3 degradation and loss to the extracellular space are not accounted for. No abnormalities in T4 and T3 uptake, T4 to T3 conversion, and electrophoretic mobility of cellular T4 and T3 binding proteins could be demonstrated in fibroblasts from two patients with congenital resistance to the action of thyroid hormone.
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