Estrogens In Pregnancy Urine Research Articles

Determination of oestrogens in pregnancy urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of three oestrogens (oestriol, oestrone and oestradiol) in pregnancy urine. Free oestrogens are extracted with chloroform from the urine sample. The phenolic group of each oestrogen in chloroform is formylated in the presence of an alkaline aqueous solution, and the resulting aldehyde is converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. In order to determine free and conjugated oestrogens, conjugated oestrogens are hydrolysed by heating in hydrochloric acid before the extraction and then treated in the same way as free oestrogens. The fluorescent derivatives are separated on a reversed-phase column, TSKgel ODS-120T, with stepwise gradient elution using an aqueous methanol-containing phosphate buffer (pH 2.2). The lower limit of detection for each oestrogen is ca. 200 fmol per 100-μl injection volume.

Studies on steroids : CLXV. Determination of isomeric catechol estrogens in pregnancy urine by high-performance liquid chromatography with electrochemical detection

A method for the determination of 2- and 4-hydroxylated estrone and estradiol in pregnancy urine by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) is described. The urine catechol estrogens were deconjugated, purified by adsorption on alumina, and subjected to HPLC-ECD. Two pairs of isomeric catechol estrogens were distinctly separated on a μBondapak C 16 column with acetonitrile-0.5% ammonium dihydrogen phosphate (pH 3.0). The amounts of these four compounds were satisfactorily determined with a quantitation limit of 1 ng using 4-hydroxy-16-oxoestradiol 17-acetate as an internal standard. The validity of the present method for the determination of urine catechol estrogens was verified by the recovery test.

Studies on immunological assay of urinary estrogens. III. A new latex agglutination inhibition reaction method.

A new, simple and rapid method for the immunological determination of estrogens in pregnancy urine is described. The principle of this method is based on the latex agglutination inhibition reaction in a solid-solid system. An antibody latex reagent (Ab-Latex) was obtained by sensitizing latex particles with anti-estriol 16-glucuronide antibody which had been raised in a goat. Estriol 16-glucuronide-bound latex reagent (E3-16-G-Latex) was prepared by the condensation of polyacrylic acid combined with estriol 16-glucuronide (E3-16-G) and lysine-latex which had been prepared from carboxylate modified latex. Hexamethylenediamine was used for the binding of E3-16-G to polyacrylic acid. The latex agglutination inhibition tests were performed on opaque glass slides. One drop (30μl) of Ab-Latex suspension followed by one drop of E3-16-G-Latex suspension was added to one drop of diluted urine sample and mixed thoroughly. The slides were rotated gently for 2 minutes, and a macroscopically visible agglutination inhibition pattern was observed in the presence of an estrogen concentration of 0.1μg/ml (as estriol) or more. The estrogen values in urine can be obtained by multiplying the sensitivity by the highest dilution factor able to give a positive reaction. This test cross-reacted not only with free estriol but also with all free and conjugated estrogens having a free hydroxy group at position 3 in the steroid ring. The urinary estrogen values obtained by this method showed a good correlation with those measured by a radioimmunoassay method (correlation coefficient ; 0.9845, regression line ; y=0.823x-0.614). When this method was compared with a semi-quantitative determination method, the hemagglutination inhibition reaction (E3 HAIR kit), the results of the two methods were in fairly good agreement.

A semi-automated fluorometric method for total estrogens in pregnancy urine.

We report a continuous-flow fluorometric method for total urinary estrogens that involves the Kober reaction, with extraction of the reaction product into dichloroethane containing trichloroacetic acid as described by Hahnel and Jones [Clin. Chim. Acta 16, 185 (1967)]. The dichloroethane extraction gives greater stability to the Kober color and sharper separation of aqueous and organic phases. The analytical system is adjusted to give maximum response to estriol 16alpha,beta-D-glucuronide, the principal estrogen conjugate in urine from late pregnancy, when calibrated with estriol standards. An initial 20-fold dilution of the sample with water increased analytical recovery of estriol conjugates from urine while maintaining adequate fluorescent response. Glucose interference was reduced by dilution and eliminated by treatment with sodium borohydride. Urinary protein up to 20 g/liter did not interfere. Total estriol, as determined by gas-liquid chromatography, comprises about 70% of total urinary estrogens in late pregnancy as measured by our continuous-flow fluorometric method. A reference range is presented based on 209 randomly collected urine specimens in which total urinary estrogens are expressed as a ratio to creatinine.

A new rapid assay of oestrogens in pregnancy urine using the substrate native fluorescence

A simple and rapid method is described for the quantitative determinations of free and conjugated oestrogens in pregnancy urine. The oestrogens are precipitated with ammonium sulphate and freed from non-oestrogenic compounds by solvent extraction. The conjugated oestrogens are hydrolysed by aβ-glucuronidase from Escherichia coli, and the total free oestrogens are extracted into ether and their fluorescence intensity at 310 nm in this solvent is determined. The method is rapid and precise for oestrogen levels at concentrations greater than 2 μg/ml (7 μmol/l). It is proposed that this method, which measures oestradiol and oestriol levels, be applied routinely to monitor feto-placental function in pregnancy. It offers advantages over other currently used assays in that less manipulative and technical skill is required to give a high level of precision and accuracy. An accurate estimate can be produced within 30–60 min of receipt of a 24-h urine specimen. Two variations of the method are also described. In one the ammonium sulphate precipitation step is omitted so as to give an even quicker assay procedure which determines conjugated oestrogens in the urine, and in the other only is determined.

Determination of estrogens in pregnancy urine by hemoagglutination inhibition reaction

A simple and rapid hemoagglutination inhibition reaction system for the estimation of estrogens in pregnancy urine is described. Results obtained by this method showed a good correlation with those measured by a conventional radioimmunoassay method and Brown's colorimetric method. Ninety-seven pregnancy urine samples from various gestational weeks were subjected to the assay, and the usefulness of this method as a screening test for high-risk pregnancy is discussed.

Effect of ampicillin administration on the excretion of twelve oestrogens in pregnancy urine.

The excretion of twelve oestrogens in urine, pooled daily from a group of pregnant women, was determined before, during and after ampicillin administration (2 g/day, for 3 days). On the second day of ampicillin administration total oestrogen excretion fell to 67% of the mean control value, oestriol excretion to 69% and that of the other eleven individual oestrogens to an average of 62% of the mean control values. In general, on the third day of treatment and on the two post-treatment days this decrease tended to be corrected. The patterns of change in the urinary levels of the individual metabolites provided no clear lead to the basic mechanism of ampicillin impairment of oestrogen excretion. However, as the drug affected all their excretion in more or less the same way as it did that of oestriol, it is possible that ampicillin interferes primarily with their enterohepatic circulation in the mother as has been established with reasonable certainty in the case of oestriol.

Gas chromatography profile of estrogens: Application to pregnancy urine

A method for the simultaneous quantitation of 7 estrogens in pregnancy urine is described. It involves enzymatic hydrolysis, extraction of free steroids, ion-exchange column chromatography and quantitation of the trimethylsilyl derivatives by gas chromatography on OV 1. Data obtained from normal and twin pregnancies and from women with anencephalic fetus or intra uterine fetal death are analysed. The sensibility of the method is about 40 μg of each estrogen by liter of urine.

A comparison of a manual and a simple automated technique for the estimation of total oestrogens in pregnancy urine

A comparison of a simple automated method for total urinary oestrogens (“oestriol”) in pregnancy with a reputedly specific manual technique showed that the results obtained using the automated method were significantly higher than those found using the manual method. This discrepancy was found to be largely, if not totally, due to losses occurring during the manual procedure, implying that the automated method gives the more accurate result.

Use of "Sephadex G-15" in an Improved Method for Estrogens in Pregnancy Urine

Abstract A simple, rapid, and reliable colorimetric method has been developed for determining total estrogens in the urine of pregnant women. It is essentially a modification of the procedure of Van Baelen et al. [J. Clin. Endocrinol.27, 1056 (1967)], in which gel adsorption is used as a purification step after enzymatic hydrolysis. Three major modifications have been made to the original procedure, whereby a single determination can be completed within 4 h. Actual manipulative time of the analysis is only 2 h, and one technician can easily handle an average of 10 cases per batch. Precision of the method is good, the coefficient of variation being less than 5%. Recovery of added estriol is better than 95%. The method can be used for measuring estrogen excretions ranging from 0.5-50 mg/24 h. We have found it very suitable as a routine assay in our laboratory.

An automated method for the determination of estrogens in pregnancy urine

Summary 1. A simple automated method is described for the determination of total estrogens in urine from pregnant subjects, using the AutoAnalyser. 2. The automated method gave good recovery and precision, and correlated well with an established manual technique. In excess of 120 samples can be evaluated daily with a throughput time of 30 minutes.

Oestrogen determination in pregnancy urine

Modifications have been made at the acid hydrolysis and the Kober colour reaction stages of the Oakey et al. 1 procedure for assaying total oestrogens in pregnancy urine. These modifications have resulted in a method whereby a single determination of urine oestriol can be carried out in 1 1 2 hours.

Automated Hydrolysis of Total Estrogens in Urine from Pregnant Women

Abstract This method for the hydrolysis of estrogens completes the automated analysis of estrogens in pregnancy urine described by Muir, Ua Conaill, and Ryan [Steroids 13, 719 (1969)]. Pregnancy urines can be processed from the sampling of urine to the recording of fluorescence intensity in 30 min, at a rate of 20 samples per hour. The presence of glucose does not decrease the apparent hydrolysis products.

Ammonium Sulfate Precipitation of Conjugated Estrogens in Pregnancy Urine: Rapid Assay and Glucose Effects

Abstract Ammonium sulfate precipitation of total estrogens in pregnancy urine effectively removes many substances that interfere with estrogen assays. From systematic study of the precipitation technique we define optimum conditions for isolating free and conjugated estrogens. Solutions of the precipitated estrogens are directly analyzed. Glucose effects on these assays are negligible. Urinary estrogens are minimally exposed to acid or alkali before analysis by the Ittrich procedure, thus protecting the labile fraction that may be significant in complicated pregnancies

An automated method for urinary oestriol determination

The addition of a predilution and two simple wash steps to the procedure of O'Connell and Muir for estimating total oestrogens in pregnancy urine results in an improvement in the recovery of oestriol from 76% to 94%. The specificity for oestriol is also enhanced, as is the correlation of results with those of a specific oestriol technique. When a fluorimetric endpoint is used with the improved procedure, the background from a urine containing insignificant amounts of oestrogens is less than 5 μg/litre. This affords the possibility that the method may also be suitable for use in non-pregnant subjects. Using the technique described it is possible for one technician to perform 70 analyses in a working day, and the result for an urgent case can be available within 2 h of receiving the urine in the laboratory.

A rapid quantitative method for the estimation of total oestrogens in human pregnancy urine

A rapid quantitative method has been devised for the estimation of total oestrogens in pregnancy urine. Acid hydrolysis and organic solvent extractions, which were necessary steps in urinary oestriol estimation by previously published methods, have been eliminated by the use of the adsorption characteristics of Sephadex G-10. In a normal working day 48 estimations can be carried out with a high degree of precision.

Removal of substances interfering with the rapid assay of estrogen in pregnancy urine.

ABSTRACT Substances interfering with the rapid assay of estrogens in pregnancy urine are largely eliminated when the estrogens are first precipitated from the urine with 70% (w/v) (NH4)2SO4. This is true for both the shortened estriol and the Ittrich total estrogen estimations. A 10-fold dilution of the urine prior to acid hydrolysis, which overcomes a marked reduction of the estrogen assay value following acid hydrolysis of undiluted diabetic urine, is usually made unnecessary when assays are carried out on solutions of the (NH4)2SO4 precipitate of the urine. In fact, a higher assay value is frequently obtained for the precipitate than for the urine from which the precipitate was prepared, when both are assayed by the dilution-acid hydrolysis technique. The precipitate can also be assayed by the Ittrich procedure or a modification thereof, even when the urine contains substances which interfere with the successful application of the procedure to the urine itself. The interfering substances in both types ...

A practical method for the fractionated fluorimetric estimation of estrogens in pregnancy urine

A method is described for the estimation of following estrogen fractions in pregnancy urine : estrone (I), estradiol-17β, 16-oxo-estradiol-17β + 16α-hydroxy-estrone (II), 16β-hydroxy-estrone (III), 16-epi-estriol + 17-epi-estriol (IV), and estriol + 16,17-epi-estriol (V). This method involves enzymatic hydrolysis, ether extraction and silicagel-chromatography for the separation of the estrogens in 5 fractions. Estradiol-17β, which is eluted in the same fraction as 16α-hydroxy-estrone + 16-oxo-estradiol-17β, can be separately determined by treating half of the eluate with a 3N NaOH solution which destructs the two Dα-ketolic estrogens. The estrogens are estimated fluorimetrically after a Kober-Ittrich reaction on the residu of the different fractions. The method is validated on the basis of criteria of specificity, accuracy and precision.

The role of ammonium sulfate precipitation in the removal of substances which interfere with enzymatic hydrolysis of conjugated estrogens in pregnancy urine.

Precipitation of the conjugated estrogens from pregnancy urine by 70% (w/v) (NH4)2SO4 was found to leave urinary substances in the supernatant which interfere with the assay following hydrolysis with enzymes (Ketodase and Glusulase). This removal of enzyme inhibitors is associated with a decrease in optimal pH for enzymic hydrolysis as well as with a reduction in the minimum amount of enzyme required to effect a maximum hydrolysis. One of the inhibitors removed is saccharolactone that may be present. An additional removal of enzyme inhibitors, with a further reduction in optimal pH, is effected by extraction of a solution of the (NH4)2SO4 precipitate by the technique of Kellie. These purification procedures result in an optimal pH for enzymatic hydrolysis equivalent to that reported for pure conjugates, and a 5- to 10-fold reduction in the minimum amount of enzyme required to effect a maximum hydrolysis. Propylene glycol, unlike its effect on phenolphthalein and pregnanediol glucuronides, has an inhibitory effect on the enzymic hydrolysis of estriol glucuronide.

A rapid method for estrogen determination in pregnancy urine

A routine method for the determination of total estrogens in pregnancy urine is described. After hydrolysis, extraction and minimal purification of the extract, the Ittrich modification of the Kober reaction is applied. Both acid, normal enzyme and rapid enzyme hydrolysis have been found useful.