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A Radioimmunoassay for Measurement of Thyronine and Its Acetic Acid Analog in Urine^*

INDER J. CHOPRA, RUBEN J. BOADO, DAVID L. GEFFNER and DAVID H. SOLOMON

Department of Medicine, University of California Center for the Health Sciences Los Angeles, California 90024

A sensitive and specific RIA has been developed to measure thyronine (T_o) in urine. The RIA used an anti-T_o antibody obtained from a rabbit immunized with a L-T_o-human serum albumin conjugate and [³H]T_o as the radioligand. The acetic acid analog of T_o (ToAc), that is the diphenyl structure with an acetic acid side-chain, cross-reacted strongly with the antibody. Relative to T_o, it cross-reacted 160% in phosphate-buffered saline, pH 7.4, and 100% in 0.075 mol/L barbital buffer, pH 8.6, containing sodium salicylate (final concentration, 8 mg/mL). The latter conditions were employed for the RIA, and the results reported thus reflect the presence of T_o and/or ToAc. 3-Monoiodothyronine, 3'-monoiodothyronine, 3',5'-diiodothyronine, and 3,5-diiodothyronine cross-reacted with the anti-T_o antibody 1.9%, 1.7%, 0.3%, and 0.2%, respectively; the cross-reactivity of other T_o derivatives and tyrosine and its derivatives was less than 0.05%.

Urinary T_o and/or ToAc excretion in 12 normal subjects averaged 16 ± 2 (±SE) µg/day (59 ± 9 nmol/day) or 14 ± 2 µg/g creatinine (5.9 ± 0.6 nmol/mmol creatinine). Treatment of urine from normal subjects with β-glucuronidase or sulfatase did not significantly alter the T_o content. Column and thin layer chromatographic studies revealed that 83% and 61%, respectively (range, 37–100%), of urinary T_o immunoreactivity was attributable to ToAc. The mean daily excretion of T_o in 20 patients with nonthyroidal illness [NTI; 22 ± 4 µg/day (82 ± 17 nmol/day)] was similar to that in normal subjects, but was elevated when expressed as nanomoles per mmol creatinine (20 ± 2; P < 0.001), because creatinine excretion was reduced in the NTI patients. The mean daily urinary To excretion in 13 patients with hyperthyroidism due to Graves' disease was slightly elevated [29 ± 6 µg/day (108 ± 21 nmol/day); P < 0.1], but was clearly elevated when expressed as nanomoles per mmol creatinine (37 ± 8; P < 0.001), again because creatinine excretion was reduced in these patients. The mean urinary T_o excretion was subnormal in 13 patients with hypothyroidism and was significantly (P < 0.005) less than that in the NTI patients regardless of the manner in which the results were expressed. Analysis of pronase hydrolysates of thyroid glands obtained at autopsy from euthyroid patients suggested that the T_o content of the thyroid approximates only 1.2% that of T₄, supporting the thesis that prior iodination of tyrosine is critical for the coupling process in the thyroid. We conclude that 1) deiodination to T_o (and/or ToAc) and its excretion in urine are the fate of a substantial proportion of the daily iodothyronine secretion in man; and 2) measurement of urinary To excretion may help differentiate patients with NTI from those with hypothyroidism.

^* This work was supported by USPHS Grant AM-16155 from the NIH.

To whom requests for reprints should be addressed.

Received November 11, 1987.