Saul L. Cohen

The conjugated estrogens: I. A new fractionation procedure suitable for large volumes of urine A: Normal pregnancy urine

Abstract The precipitation of the estrogen conjugates with ammonium sulphate has permitted for the filtration through Sephadex of large volumes of pregnancy urine equivalents (up to 170 l). Such a filtration together with assay of all the estrogens instead of just the classical three, results in the attainment of six peaks, instead of just two as described by Beling. Small amounts of estrogens, not including the classical three, seem to be strongly adsorbed onto the column, and require for their elution very large volumes of water. These “estrogens” can be detected by thin-layer chromatography, and on this basis are grouped into fractions. It requires a total volume of about 300 l to effect the elution of all the estrogens in batches of 80–170 l normal pregnancy urine. The total conjugated estrogens of this urine have been differentiated into 20 separate fractions by the ammonium sulphate precipitation and Sephadex filtration techniques. By thin-layer chromatography, there are one to as many as eight (usually four to five, with considerable overlapping) different estrogens in each fraction. Five of the fractions appear sufficiently pure to permit crystallization of the conjugated estrogens.

A function for estriol during human pregnancy—a hypothesis

It has been hypothesized that large amounts of estriol (E3) are produced during human pregnancy to ensure a quiescent uterus during prelabour pregnancy by combining with most of the myometrial nuclear receptors, leaving an inadequate number for a stimulatory estradiol (E2) concentration. It is further hypothesized that the amount of E3 formed is controlled by the amount of E2 present. During labour this control is lost, which together with an increased E2 (plus or minus a simultaneous drop in E3) production permits labour. Two of the seven pieces of evidence offered in support of this hypothesis were carried out in the authors laboratory. These are: 1. Urinary assays by two methods, one for total estrogens and one for the fractionated classical estrogens revealed that while the ratio (formula; see text) varies from patient to patient, for any one patient it remains markedly constant, especially during the second half of pregnancy. 2. Pieces of myometrium removed at cesarean delivery and assayed for their nuclear estrogen content revealed an E3/E2 ratio of 1.7 when the cesarean was an elective one (and therefore with a quiescent uterus) but reduced to 0.65 when the cesarean was performed after labour had started. The relationship between these two pieces of evidence and five from the literature with the hypothesis are discussed.

The changing estrogen content of the nuclear fraction of human myometrium during labor

The cytosol and nuclear fractions were prepared from 32 pieces of myometrium obtained from 20 elective cesarean sections (regarded as typical of quiescent pregnancy (P)) and 12 emergency cesareans (performed after labor had started and therefore taken as typical of labor (L)). The nuclear fraction was purified by standard procedures. All protein-bound estrogen was released from the nuclear fractions, and the released estrogen simultaneously solubilized by denaturation with ethanol. The estriol (E3) and estradiol (E2) content of the alcohol solutions were assayed by radioimmunoassay with highly specific antisera for E3 and E2. In the L group, average E3 content was slightly (not significantly) lower, and average E2 content was significantly (P less than 0.005) higher, than in the P group. The E3/E2 ratio decreased dramatically (P less than 0.001) during this change from P to L.