Thyroid Function Testing

Measurements of Serum T₄

Complications due to Protein Binding – 99.97% of T₄ is protein-bound in serum, but only free T₄ has metabolic effects

thyroid binding globulin (TBG) is made by the liver – production Ý in pregnancy (due to estrogen), ß in nephrosis

free T₄ is carefully regulated by TSH – if Ý binding hormone, more T₄ will be secreted to normalize free T₄ Þ Ý total T₄

total T₄ can estimate metabolic status (hyporthyroid, euthyroid, thyrotoxic) only if binding protein levels are normal

Tests to Measure Serum T₄:

(1) Protein Bound Iodine Test – measures iodinated proteins; T₄ accounts for most of these – test » total T₄

normal levels are 5-12 m g/dl

does not indicate free fraction of T₄, so Ý TBG Þ falsely high results; ß TBG Þ falsely low results

(2) Radioimmunoassay – measures total T₄ (protein-bound and free) like #1 above

(3) Dialysis Equilibrium Test – best test for free T₄, but technically difficult and expensive – no longer used

serum incubated with known amount of radiolabelled T₄ – TBG binds a certain fraction of this T₄
measure fraction of radiolabelled T₄ that equilibrates past dialyzing membrane – this is free fraction of T₄

if total T₄ is measured, can calculate free T₄ by multiplying (% free) x (total T₄)

(4) Resin T₃ Uptake (RT₃U) Test – currently the most useful test – indirectly measures free T₄

serum incubated with known amount of radiolabelled T₃ (easier than T₄ for technical reasons) that binds TBG's free binding sites (i.e., those not already bound to T₄)

a T₃-binding resin is added, which binds free T₃ that is left over after all available TBG binding sites are occupied; therefore, radioactive resin-bound T₃ indicates amount of thyroid hormone that did not bind TBG (normally 35-40%)

RT₃U is directly proportional to free T₄ levels (at a given TBG)

Note: RT₃U is also inversely proportional to the amount of TBG in the serum; more TBG present means less T₃ will be free to be picked up by the resin, and vice versa

results vary from day to day – therefore do a ratio test: more consistent value, expressed as %

ratio test: resin T₃ uptake is divided by a normalized value from pooled human serum; normal value = 0.8-1.2

How can this be used to estimate free T₄? – multiply total T₄ by RT₃U to yield Free T₄ Index (» free T₄)

this works because the number of labeled free T₄ is proportional to the number of unbound sites on thyroid binding protein!!

Condition	TBG	Total T₄		RT₃U		Free T₄ Index
Normal	Normal	Normal	X	Normal	=	Normal
Pregnant	High	High	X	Low	=	Normal
Hepatic/Renal Disease	Low	Low	X	High	=	Normal
Hyperthyroid	Normal	High	X	High	=	High
Hypothyroid	Normal	Low	X	Low	=	Low
Hyperthyroid and Pregnant	High	Very High	x	Normal	=	High

Note: This test cannot distinguish hyperthyroidism from Ý binding protein (or hypothyroidism from ß binding protein)

remember, it is the number of free binding sites on TBG that is measured, not the actual hormone levels

(5) Free-T₄ Radio-Immunoassay – directly measures free T₄ using a high-affinity antibody

use antibody-coated test tube or glass beads
this test is not yet in common use

Measurements of Serum T₃

T₃ is T₄ with the 5’ iodine atom removed, but its action is 4x more potent than T₄ – it is 99.7% protein bound in serum

The thyroid only secretes 20% of T₃; the rest is made by the enzyme 5’ monodeionidase in the liver and kidneys

Tests to Measure Serum T₃

Same as for T₄ (#2,3,4 above) – best clinical measurement is Free T₃ Index (= total T₃ x RT₃U, #4 above)
there is currently no way to measure free T₃ directly by radioimmunoassay (#5 above)

Measurements of Serum Thyroid Stimulating Hormone (TSH)

TSH secretion is tightly controlled by feedback inhibition from T₃ and T₄, so TSH is inversely proportional to thyroid function

therefore, Ý TSH Þ hypothyroid, and ß TSH Þ hyperthyroid (i.e., Grave’s Disease)

Radioactive Iodine Uptake

This test indicates degree of activity of thyroid itself, not just amount of thyroid hormone secreted

useful to distinguish excessive thyroid hormone synthesis from leakage from inflamed thyroid

Test Protocol – after oral tracer dose of radioactive iodine, measure 24-hour uptake into thyroid

measurement made via thyroid scan – a radiograph of the thyroid
if nodule is "hot" (takes up much iodine), it is unlikely to be cancerous; if "cold", cancer cannot be ruled out

Factors that Invalidate Test

abnormally high total body iodine content – will dilute out radioactive iodine tracer Þ low uptake by gland

drugs that block thyroid hormone synthesis (Propylthiouracil, etc.) Þ iodine not used so it leaves gland immediately