Direct determination of free triiodothyronine (T-3) in undiluted serum by equilibrium dialysis/radioimmunoassay (RIA)

We have devised a practical, sensitive, and reliable assay for measurement of free T-3 concentration in serum. The assay employs a convenient and disposable plastic equilibrium dialysis cell and a buffer that resembles the in vivo biochemical environment (Nelson JC, Tomei RT 1988 Clin Chem 34:1737). A 200-mu l aliquot of serum was dialyzed against 2.4 mt buffer at 37 degrees C for 18 +/- 2 h and T-3 Was quantified by RIA of about 1.0-mL aliquot of the dialysate buffer. The detection threshold of the RIA approximated 2 pg/ml permitting accurate measurement of >200 pg/dL of free T-3 directly. Serum specimens that contained less free T-3 Were spiked with 200 ng/dL of nonradioactive T-3 prior to dialysis. Free T-3 in the dialysate of these samples was divided by total T-3 in serum (after spiking) to determine percent free T-3. Free T-3 was calculated by multiplying percent free T-3 and serum total T-3 (before spiking). Free T-3 concentration (pg/dL) did not differ appreciably in a serum pool when tested both with and without spiking with exogenous T-3. The between assay coefficient of variation of three specimens tested over an 8-month period averaged 20%. Serum free T-3 concentration (pg/dL) was [mean +/- SD (rt), range, p] [293 +/- 12 (39), 154-140] in normal subjects. It was significantly increased [742 +/- 87 (13), 525-1700, p < 0.001] in hyperthyroidism and significantly decreased in nonthyroidal illness [NTI, 138 +/- 26 (9), 53-320, p < 0.001], cord blood serum [124 +/- 7.5 (11), 93-353, p < 0.001], and third trimester of pregnancy [214 +/- 26 (8), 93-253, p < 0.02]. Serum free T-3 concentration varied widely in hypothyroidism 274 +/- 92 (10), 10-923, NS]. Conclusions: We have described a practical method and initial results of direct measurements of free T-3 concentration in health and disease.