Free and Conjugated Estrogens in Blood Plasma during Human Pregnancy^*

Abstract

The concentrations of the free, glucosiduronate, and sulfate forms of estrone (E,), estradiol17p (E2), and estriol (E3) in maternal and umbilical cord blood are reported. Phenolsulfatase and @-glucuronidase hydrolyses followed extraction of the free ether-extractible estrogen. There was more El and Ez in the conjugated forms in the maternal than in cord plasma. El sulfate (4.1 rg) was the major estrogen S everal studies describing estrogen levels in blood have been published recently (Diczfalusy and Magnusson, 1958;Roy and Mackay, 1962; Maner ef al., 1963 ;Aldercreutz, 1964). These reports have evaluated the total amounts of estrone, estradiol-l7P, and estriol rather than differentiating between free and conjugated steroids. Recent reports from this laboratory describe a comparison of the conjugation of estriol in umbilical cord and in the maternal peripheral blood plasma . A preliminary report described the conjugation of estrone and estradiol-l 7p in both cord and maternal blood .The present report gives details of methodology and results of quantitation of estrone, estradiol-17& and estriol in plasma in the free form and after liberation by hydrolysis with @-glucuronidase and phenolsulfatase (Mylase P) in a larger series. Conjugates of estrone and estradiol-17P were found in larger quantity in the maternal blood than in cord blood. No estrogens were found in the red cells. Apparatus and Column ConditionsThe gas chromatograph used in this work was (Glowall Corp. Model A-110) equipped with a Lovelock detector using radium foil. The column, a glass coil (183 cm x 4 mm), was packed with a combination of 10% QF-1 (fluorosilicone polymer, Dow Corning) and 5 % L-45 (methylsilicone polymer, Dow Corning) on Gas-Chrom Z, 80-100 mesh (Applied Science). The substrates were coated on the support in one application other than E8 sulfate (4.4 pg 100 ml) found in the maternal blood. Both free E, and El were found in higher concentrations that their glucosiduronates.Results for concentrations of the ES forms agreed with the previous work (J. C. Touchstone, J. W. Greene, Jr., R. C. McElroy, and T. Murawec [1963], Biochemistry 2, 653). No estrogen was found in the red cells by the present method. by dissolving them in methylene chloride (5-6 volumes x the weight of support) and evaporating the solvent with constant stirring after addition of the support. The packing was dried in a vacuum oven and packed in the glass coil under a vacuum. The columns were conditioned at 270" for 2 days with argon flow at an inlet pressure of 10 psi. Operating conditions were: column and detector temperature, 250 " ; flash temperature, 270"; inlet pressure, 30 psi; and voltage, 1000. Samples were dissolved in 25 p1 of t-butyl alcohol and 2 to 4-pl aliquots were injected.Preparation of Red Cells and Blood Plasma Extracts. Heparinized blood was collected from the antecubital vein of women in the third trimester of pregnancy. Blood was milked from the umbilical cord at the time of separation from the fetus and pooled. Pools of 100 ml or more were use...