Cholecystographic agents and sulfobromophthalein inhibit the binding of L-thyroxine to plasma membranes of rat hepatocytes.

Cholecystographic agents and sulfobromophthalein (BSP) cause a major discharge of labeled T4 from the liver in man in vivo. In the present study we sought to determine if this discharge is partially due to inhibition of T4 binding to plasma membrane sites. Plasma membranes were isolated from hepatocytes of female Sprague-Dawley rats, and 5'-nucleotidase levels were measured to demonstrate plasma membrane viability. Specific binding of T4 (Ka, 1.01 X 10(8) M) was confirmed by displacement of labeled T4 by unlabeled hormone (10(-10)-10(-5) M). Displacement of labeled hormone was also produced by addition of tyropanoate, iopanoate, ipodate, or BSP. At 5-mM concentrations of inhibitor, the Ka for T4 declined to 4.00 X 10(7) M with BSP, 5.07 X 10(7) M with ipodate, 5.62 X 10(7) M with tyropanoate, and 7.43 X 10(7) M with iopanoate. Thus, a portion of the discharge of hepatic T4 after administration of these agents may be due to competitive inhibition of binding to plasma membrane sites.

Effects of cholecystographic agents and sulfobromophthalein on binding of thyroid hormones to serum proteins.

A number of interactions between thyroid hormones and cholecystographic agents have previously been demonstrated. In the present study we show that cholecystographic agents also interfere with the binding of thyroid hormones to serum proteins. A commercial kit (Tri-Tab) was used in which the uptake of labeled hormone from serum by a silicate adsorbent tablet is measured. In the presence of cholecystographic agents or sulfobromophthalein (BSP), the amount of labeled hormone bound to adsorbent increased in a dose-dependent fashion, reflecting displacement from protein-binding sites. The order of potency was BSP greater than ipodate greater than iopanoate greater than tyropanoate. Displacement of hormone was confirmed by a second methodology in which graded amounts of unlabeled T4 were added to the system. This allowed a Scatchard analysis to be performed for binding sites on T4-binding globulin. The cholecystographic agents and BSP caused displacement of the Scatchard slopes, again demonstrating interference with binding to serum protein sites. A method is described in which the change in Scatchard slope produced by an inhibitor is employed to compute the association constant between T4-binding sites on T4-binding globulin and the inhibitors. The values were: BSP, 14.6 X 10(3) M-1; ipodate, 4.7 X 10(3) M-1, iopanoate, 2.2 X 10(3) M-1; and tyropanoate, 0.1 X 10(3) M-1. Because of these relatively low values and the rapidity with which these agents are normally cleared from serum, it seems likely that effects on free hormone levels would be transient and of small magnitude during routine cholecystography. Also, ipodate, in the 1 g/day dose that has been employed experimentally to treat hyperthyroidism, should have a negligible effect on protein binding. On the other hand, when high levels of these compounds are used in experimental settings to study other aspects of thyroid hormone metabolism, changes in protein binding can occur and confound interpretation of results.

Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines.

The enzymic 5'-deiodination of 3',5'-di-iodothyronine and 5-deiodination of 3,3',5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3',5'-Di-iodothyronine was a competitive inhibitor of 3,3',5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3',5-tri-iodothyronine was a competitive inhibitor of 3',5'-di-iodothyronine 5'-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.

Inhibition of hepatic binding of thyroxine by cholecystographic agents.

Subsequent to studies indicating that cholecystographic agents and sulfobromophthalein (BSP) inhibit uptake of thyroxine (T(4)) by rat liver slices, the effect of such compounds on hepatic storage of T(4) in man has been examined. After intravenous administration of [(125)I]T(4) to five normal subjects, hepatic radioactivity, estimated by external gamma counting, rose to a peak in approximately 4 h and then declined in parallel with serum radioactivity. When a 6-g dose of the cholecystographic agent, tyropanoate (Bilopaque), was administered orally 3 d later, estimated hepatic extravascular radioactivity fell 50-60% within 4 h and then rose toward the pretyropanoate value. Concomitant with the fall in hepatic radioactivity, serum radioactivity rose 57-70%, as did stable T(4) levels in serum, suggesting that hormone discharged from the liver entered the serum. Both uptake of T(4) and discharge by tyropanoate were much less in two patients with liver disease. Ipodate (Oragrafin), 12 g orally in two subjects, caused much smaller changes in hepatic and serum radioactivity. However, ipodate also caused a doubling of the percent free T(4) in the serum as judged by equilibrium dialysis, and the ratio of hepatic radioactivity to free [(125)I]T(4) in serum declined markedly after ipodate, similar to the fall in hepatic:serum (125)I ratios after tyropanoate. BSP, 20 mg/kg i.v. in three subjects, caused a smaller change; the decline in hepatic T4 content averaged 19%. We conclude that these organic anions, tyropanoate, ipodate, and BSP, all can displace T(4) from the liver. The results imply a link between T(4) transport and organic anion transport, and indicate a mechanism for altering hepatic T(4) content in man that could be the site of physiologic regulation or of disease. A method is described whereby analysis of the change in hepatic and plasma radioactivity after tyropanoate could be employed to estimate hepatic T(4) content in diverse clinical circumstances.