Serum free T4, T3, rT3, 3,3'-diiodothyronine and 3',5'-diiodothyronine measured by ultrafiltration

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Abstract
A simple and accurate method for estimation of the free fractions (FFT) of T4 [thyroxine], T3 [triiodothyronine], r[reverse]T3, 3,3''-diiodothyronine (3,3''-T2) and 3'',5''-diiodothyronine (3'',5''-T2) in serum is presented. The method is based on ultrafiltration of serum pre-incubated with tracers of high specific activity, followed by purification of the ultrafiltrate on small Sephadex columns. The addition of tracer only dilutes serum negligibly (about 5%) and the ultrafiltration procedure only removes about 7% of the volume of serum, thus probably not disturbing the equilibrium between the free and protein bound fraction of iodothyronine. Progressive reduction of tracer to less than 10% of the amount usually used did not reduce the FFT of any of the iodothyronines. Addition of T4 to serum led to an increase of all FFT except that of 3'',5''-T2. Thus FFT of T4, T3, rT3 and 3,3''-T2 primarily is determined by the amount of T4 present in serum and significant amounts of these iodothyronines are bound to TBC [thyroxine binding globulin], whereas 3'',5''-T2 possibly primarily is bound to albumin. The median FFT of T4, T3, 3,3''-T2 and 3'',5''-T2 in serum from euthyroid subjects (n = 38) was: 0.030, 0.29, 0.14, 1.10 and 1.07%, respectively. The corresponding median free concentrations in pmol/l were: 30, 4.79, 0.59, 0.44 and 0.77, respectively. Pregnant women in 3rd trimester had normal levels of free T4, free T3 and free rT3, whereas the median free 3,3''-T2 was reduced in contrast to elevated median free 3'',5''-T2. Using the present method free T4 and free T3 discriminated almost completely hyper- and hypothyroid patients from controls, whereas some overlap to normal range was found concerning free rT3, free 3,3''-T2 and free 3'',5''-T2. Patients with liver cirrhosis or chronic renal failure (GRF) had profound changes in total serum concentrations of all iodothyronines studied, possibly due to the somatic disease. Despite this, almost all had unchanged free T4 and free T3 levels indicative of euthyroidism. The median free rT3 and free 3'',5''-T2 were elevated in cirrhosis but unchanged in GRF, whereas median 3,3''-T2 was unchanged in both groups.

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