T4 and T3 have been measured by RIA in 10-12-day-old rat embryo-trophoblasts, and in 13-20-day-old embryos and placentas, as well as in a few samples of amniotic fluid. Both T4 and T3 were measured after extraction of the samples with ethanol, purification by paper chromatography, anion exchange resin, or both. T4 and T3 could be shown in all samples studied. The amounts of T4 and T3 per conceptus and their concentrations were higher in embryo-trophoblasts and placentas than in 13-18-day-old embryos. The concentrations of T4 and T3 remained fairly constant in the embryos until day 19, when they appeared to increase. The molar ratios of T4 to T3 were 1.4, 8.5 and 103 for embryos, placentas and maternal plasma, respectively. These data show that, for at least one mammalian species, embryonic tissues are provided with T4 and T3 from the earliest date studied, namely 4 days after uterine implantation, and well before onset of thyroid function, which in the rat starts after 17 days gestational age. Such a result suggests that statements denying a possible role of thyroid hormones in early embryogenesis ought to be reconsidered.

To determine whether T4 has an intrinsic effect at the pituitary level, it would be important to block conversion of T4 to T3 completely. We have attempted to achieve this with iopanoic acid (IOP), a radiographic contrast agent. We have then measured in the same animals the effects of such treatment on the conversion of T4 to T3 or on the deiodination of T3 and on the pituitary response to a dose of T4 or T3. Plasma TSH levels and pituitary GH content were measured as biological end points. Thyroidectomized rats were injected with a single dose of T4 (1.7 micrograms/100 g BW) labeled with [125I]T4 (Exp A) or with a single dose of T3 (0.33 microgram/100 g BW) labeled with [125I]T3 (Exp B) and treated with IOP or solvent. Animals of Exp A were killed 24 h after iodothyronine injection and those of Exp B were killed 4, 12, and 24 h after injection of the iodothyronine. The concentrations of [125I]T4 and [125I]T3 were measured in several tissues, including the anterior pituitary, after extraction and paper chromatography and quantified with the aid of 131I-labeled markers added in vitro. Plasma and pituitary T3 and T4 plasma TSH, and pituitary GH were measured by specific RIAs. Results show that treatment with IOP markedly inhibits the conversion of T4 to T3 and the deiodination of T3. In IOP-treated thyroidectomized rats, the injection of T4 results in little, if any, effect at the pituitary level, despite an almost 3-fold increase in the percentage of injected T4 found in the gland. Treatment with IOP does not inhibit the effects of a T3 dose; if anything, they appear to be enhanced. It is concluded that, as assessed from biological responses involving the anterior pituitary, a dose of T4 has little if any effect other than that which can be attributed to the T3 generated from it.

Female rats were placed on a low iodine diet (LID) or LID supplemented with KI. They were mated 3-6 months later. Maternal and embryonic tissues were obtained both before the onset of fetal thyroid function, at 11 and 17 days of pregnancy, and at 21 days of gestation. T4 and T3 concentrations were measured by RIA. T4 concentrations were very low in the plasma, liver, and lung of LID dams and in all embryonic samples obtained from such mothers, namely 11-day-old embryotrophoblasts, 17-day-old placentas and embryos, 21-day-old placentas, embryos, plasma, liver, lung, and carcass (whole embryos minus the trachea, thyroid, blood, liver, and brain). T3 was low in 17-day-old placentas and embryos and in all fetal tissues obtained at 21 days of gestation from LID dams. These results show that when iodine deficiency is severe enough to result in very low maternal plasma T4 levels, embryonic tissues are deficient in T4 and T3 both before and after the onset of fetal thyroid function. This finding might be relevant to the etiopathology of human iodine deficiency disorders.

Female rats were killed 15 days, 2 months, and 4 months after surgical thyroidectomy that was followed by injection of 100 microCi 131I. The concentrations of T3 and T4 were measured in tissues (liver, kidney, brain, heart, and hindleg muscle) specific RIAs. Results were compared to those found in intact rats. Thyroidectomy resulted in severe hypothyroidism by 2 and 4 months after the operation, as assessed by undetectable levels of T4 and T3 in unextracted plasma, high circulating TSH, hypothermia, stasis of body weight increase, and depletion of pituitary GH content. Concentrations of T4 and T3 in plasma, as determined after extraction and concentration, were very low, being less than 5% of the normal value by the earliest observation period (15 days). In contrast, although tissue concentrations and total organ contents also decreased after thyroidectomy, they were still clearly detectable 4 months after thyroidectomy. The rates of decrease of T4 and T3 concentrations in most tissues were markedly slower than expected from their rapid decrease in plasma. Some tissues still contained 20% of the normal level 2-4 months after ablation of the thyroid. Tissue levels of thyroid hormones were hardly detectable in rats thyroidectomized 6 months before, having decreased in most tissues to less than 5% of the normal value. Several animals from this group had died. It is concluded that tissues from severely hypothyroid thyroidectomized rats may contain higher concentrations of T4 and T3 than previously thought. The idea that thyroid hormone is not essential for life, based on the assumption that thyroidectomized animals survive without thyroid hormones, might have to be reevaluated.

Previous results are contradictory regarding the concentration of thyroxin in human milk. Using a sensitive radioimmunoassay, we have found a lack of parallelism between the standard curve for thyroxin and the curve for serial dilutions of whole human milk, skimmed milk, or ethanol extracts of milk. Nonspecific binding also indicated the presence of analytical artifacts. Thus we have separated thyroxin from other milk components by means of a strongly basic Bio-Rad anion-exchange resin with quaternary ammonium exchange groups attached to a styrene divinyl benzene copolymer lattice, radioimmunoassaying the fractions eluted with an equivolume mixture of acetic acid and water. Parallelism with the standard curve was good, and results were the same whether or not the resin eluate was further purified by paper chromatography. The range of thyroxin concentration in 21 samples of human milk was 0.29-2.00 micrograms/L (mean 0.71, SD 0.40, microgram/L). Such concentrations are unlikely to afford protection to the developing brain of a breast-fed athyreotic baby, as previously claimed.