Free Serum Thyroxine:
Its role in diagnosis of thryroid disease.
Author:
I-Wen Chen, Ph.D., Professor
The thyroid hormones, triiodothyronine (T3) and thyroxine (T4) are highly bound to the plasma proteins, thyroxine binding globulin, albumin and pre-albumin. Only the unbound (free) portions (approximately 0.03% in the case of T4) are biologically active and responsible for the regulation of thyroid function through the pituitary feedback mechanism. In patients with no underlying systemic illness or condition known to alter binding proteins, the total serum T4 concentration is a specific and sensitive index of thyroid function. However, a change in the concentration or binding of plasma proteins can alter the total T4 concentration dramatically while the concentration of free T4 remains relatively constant. Under these circumstances, the total measurement will be misleading and a free T4 concentration is needed to accurately assess the hormonal status of the patient. Conditions which can lead to altered thyroid hormone binding include the following:
- Burns
- Critical illness
- Malignancy
- Nephrotic syndrome
- Trauma
- Cirrhosis
- Hepatic abscess
- Malnutrition
- Pregnancy
- Surgery
The American Thyroid Association (ATA) is currently recommending that a free T4 estimation and a sensitive (2nd generation or better) thyrotropin (TSH) assay be used to detect thyroid disease (1). Direct or indirect methods can be used to determine the free serum T4 concentration.
Direct Determination of Serum Free T4 Concentrations
The most accurate and reliable methods presently available for directly measuring free T4 utilize equilibrium dialysis and ultrafiltration methods. Both are cumbersome, technically demanding, difficult to automate and, therefore, expensive and used primarily as reference methods (2).
Indirect Estimates of Free Serum T4 Concentrations
Three procedures for the indirect determination of free T4 are available. They are the index, one-step, and two-step methods.
Index Method - This is the historical method which utilizes total T4 and so-called T-uptake measurements to calculate a free thyroxine index (FTI).
One-step (Analog) Method - This method is based on the assumption that structurally modified and labeled analogs of T4 will not bind to serum thyroid hormone binding proteins but will compete with free T4 for binding to the T4 antibody introduced in the assay.
Two-step (Immuno-Extraction) Method - Free T4 in patient serum is removed by binding to T4 antibody which is attached to a solid phase. The serum is then removed and the remaining antibody sites on the solid phase are back-titrated with a labeled thyroid hormone in order to obtain a measure of the amount of free T4 extracted from the patient's serum. Provided the amount of free T4 does not exceed 5% of the total, results will provide a good estimation of the free T4 concentration.
Effects of Thyroid Binding Proteins on Indirect Estimates of Free T4
Patients with extreme abnormalities in thyroid hormone binding give erroneous results when the index and one-step methods are used. Such patients include those with the following:
- abnormal thyroxine binding globulin or prealbumin
- familial dysalbuminemia
- T4 to T3 autoantibodies
Such interferences cause relatively minor effects in the two-step method because there is no interaction between the thyroid binding proteins in the patient's serum and the labeled thyroid introduced during the assay (3).
In our laboratory, we have shown that the free T4 value obtained using the two-step assay correlated well with the direct equilibrium dialysis method in a patient with autoantibodies to T4 and T3 (3), while spuriously high values were obtained with several one-step assay systems. However, the two-step method can produce abnormal high or low free T4 concentrations in patients with severe non-thyroidal illness (4).
Conclusion
Since free T4 is the active form of T4, it appears that measurement of free T4 is likely to be a better indicator of thyroid function than is total T4 especially in patients with protein abnormalities. Since the most accurate methods presently available for measurement of serum T4 - equilibrium dialysis and ultrafiltration - are cumbersome, expensive and technically demanding, we have evaluated several indirect methods, finding that the two-step method correlates well with the reference methods. A two-step method is currently under development.
References
- Surks MI, Shopra IJ, Mariash CN, et al. American Thyroid Association guidelines for use of laboratory tests in thyroid disorders. J Am Med Assoc 1990;163:1529-32.
- Nelson JC, Tomei RT. Direct determination of free thyroxine in undiluted serum by equilibrium dialysis/RIA. Clin Chem 1988;34:1737-44.
- Chen I, Sperling M, Canfield P, et al. Interference by autoantibodies to thyroxine and triiodothyronine in their measurement by automated immunoassay systems. Abstract, 9th Int Congress of Endocrinology, 1992, 236.
- Hay ID, Bayer MF, Kaplan MM, et al. American Thyroid Association assessment of current free thyroid hormone and thyrotropin measurements and guidelines for future clinical assays. Clin Chem 1991;37:2002-8.