The next problem is the nature of the radioactive material in the tissues and whether this is different in uterus and vagina from that in other tissues. Immature female rats were injected intravenously with 0.1 µg. of tritiated estradiol; the uteri (including cervices) were removed and pooled from 24 animals after 2 hours and from 36 animals after 6 hours. The pooled uteri were homogenized in ten times their weight of water in the presence of 5 µg. each of estradiol, estrone, and estriol as carrier. Four volumes of absolute ethanol were added, and the precipitated protein was removed by centrifuga-tion and washed several times with absolute ethanol. The combined alcoholic solutions were evaporated at reduced pressure and the residue partitioned between ether and water. Aliquot portions were taken for counting at all stages, and the results are summarized in Table I, expressed as percentage of the total radioactivity in each homogenate. It is clear that, in uterus, very little radioactive material is bound to the alcohol-insoluble protein and that relatively little is in a water-soluble form. The picture is quite different in liver taken at 2 hours, when about half the radioactive material is bound to the protein precipitate and, of the unbound protein portion, about three-fourths is in a water-soluble form becoming ether-soluble after acid hydrolysis.

Table I. Fractionation Of Radioactive Tissues After Single Intravenous Injection Of 0.1 µG. High-Level Estradiol In Saline*

* As per cent of total radioactivity in homogenate.

When the ether-soluble fractions, representing most of the radioactive material from the 2-hour and 6-hour uteri, were chromatographed on paper in a slightly modified Bush B-S system, practically all the radioactivity moved with the estradiol fraction (Table II). Elution and rechromatography in a Bush B-3 system confirmed this identification. There was a very small amount of activity in the estrone fraction from the 6-hour uteri, which has yet to be rechromatographed and identified with certainty. In the same table are the results with the ether fraction from an experiment in which 0.1 µg. of estrone was administered intravenously. Although the latter is a single preliminary experiment which should be considered as only tentative, it suggests that administered estrone may be converted to a variety of products. It is interesting to note that Gallagher (3) has reported recently that the common urinary estrogen metabolites appear to arise from estrone rather than from estradiol.

Table II. Chromatography Of Ether-Soluble Fraction From Uterus* (0.1 µC. Steroid, I.V.)

*As per cent of activity recovered. Chromatography in modified Bush B-5 system in presence of carrier estradiol, estrone, and estriol.

Table III. Fractionation Of Radioactive Tissues After Seven Daily Subcutaneous Injections Of 0.05 Ug. High-Level Estradiol In Sesame Oil*

* As per cent of total radioactivity in homogenate. Tissues taken 24 hours after the last injection.

A somewhat similar pattern was seen when 0.05 µg. of estradiol in sesame oil was administered daily for 7 days with the uteri and livers taken 24 hours after the last injection (Table III). On fractionation as previously described, again there was practically no binding of the steroid to the alcohol-insoluble protein of uterus and the major portion of the radioactive material was soluble in ether. In the liver, on the other hand, a substantial amount of radioactive substance was bound to protein, although in this 7-day experiment relatively little water-soluble metabolite was present. On paper chromatography, most of the radioactivity of the ether soluble fraction from uteri moved with estradiol, although in this case, there appeared to be two other significant components, one more polar than estradiol and one less polar (Table IV). Chromatography of the ether -soluble material from liver gave no predominant radioactive fraction; further work is necessary for the identification of the substances present.

Table IV. Chromatography Of Ether-Soluble Fraction* (7 X 0.05 µG. Estradiol, Subcutaneously In Oil)

* As per cent of activity recovered. Chromatography in modified Bush B-5 system in presence of carrier estradiol, estrone, and estriol.

Thus, it is clear that the chemical nature of the radioactive material present in rat uterus following the administration of tritiated estradiol differs considerably from that present in liver, and the uterine steroid would appear to be predominantly unchanged estradiol under all the conditions investigated.

The final and, perhaps most important, question we should like to answer is whether there is a selective intracellular localization of the radioactive steroid in the mitochondria, microsomes, nuclei, cytoplasm, or perhaps cell membranes. At present, we can merely pose the question and state that we are investigating the problem by means of our tritiated estrogen, using both fractionation and autoradiographic techniques.