open access

https://doi.org/10.11501/3137290

https://hdl.handle.net/2115/51519

Solid-Phase Extraction of Estrogens from Bovine Blood Plasma for their Peripheral Detection by Radioimmunoassay

日本語

1998-03-25

Hokkaido University

50p.

A simple purification and separation method for estrogens in bovine peripheral blood with solid-phase extraction was established for potential contribution to research on reproductive physiology in cattle. As a preliminary experiment on elution of steroids in the solid-phase extraction with a small reversed-phase cartridge, 5 unconjugated steroids (estrone, estradiol-17β,) testosterone, androstenedione and progesterone) and 1 conjugated steroid (estrone sulfate), all important in female reproduction, were analyzed by high performance liquid chromatography (HPLC) with a high pressure reversed-phase column. The elution patterns suggested the possibility of separately collecting unconjugated and conjugated estrogens as well as estrogens and progesterone with a small reversed-phase cartridge, although this would not be possible for estrone and estradiol-17β, and estrogens and androgens. Whether unconjugated and conjugated estrogens added to bovine plasma can be extracted and purified by solid-phase extraction with a small reversed-phase cartridge has been investigated. According to results of HPLC, methanol was used as elution solvent in the following. First, 3H-labeled estrogens added to bovine plasma at various reproductive stages were recovered by solid-phase extraction with applied volume to ascertain whether estrogens in the plasma are quantitatively retained in the cartridge. Secondly, unconjugated and conjugated estrogens retained in the cartridge were eluted with a stepwise gradient of methanol concentration to determine an appropriate concentration of methanol for the elution of each estrogen. Thirdly, study was made as to whether unconjugated and conjugated estrogens are separately recovered with a stepwise gradient of methanol concentration. All estrogens were highly and quantitatively retained in the cartridge irrespective of the reproductive stage or applied plasma volume. The elution pattern of each estrogen with the stepwise gradient was consistent with that in HPLC. When estrogens retained in the cartridge were eluted by the stepwise gradient with 40 and 70% (v/v) methanol, estrone sulfate was eluted with 40% methanol, followed by estrone and estradiol-17β with 70% methanol. Thus, estrogens in bovine blood plasma can be quantitatively extracted by solid-phase extraction with a small reversed-phase cartridge. Unconjugated and conjugated estrogens retained in the cartridge appeared to be separately recovered. Endogenous estrogens were extracted from bovine blood samples with a reversed-phase cartridge and measured by radioimmunoassay(RIA) to demonstrate the reliability of the total assay system. First, the influence of applied plasma volume on the assayed value was examined and appropriate plasma volume for the assay was determined. Impurities affecting the reaction of estrogen on RIA were characterized and the influence of bovirle plasma component on the extraction of endogenous estrogen was investigated. The recoveries of exogenous estrogens by solid-phase extraction and RIA were examined to verify assayed values. Estradiol-17β was isolated from the same blood samples by solid-phase extraction as well as liquid-liquid extraction followed by distribution by liquid chromatography as a validated extraction and purification method. Estradiol-17β in each extract was measured by RIA and the values were compared. Endogenous estrogen was found to be adsorbed quantitatively in the cartridge, and unconjugated and conjugated estrogens to be separately eluted by stepwise gradient with 40 and 75% methanol. Although RIA values declined with increase in applied plasma volume, unconjugated and conjugated estrogens included in the bovine plasma could be quantitatively measured for the applied plasma volume of 2 ml or less. Concentrations could be almost precisely determined by correcting the assayed values with recovery in known samples, even if estrogen levels were so low that more than 2 ml of plasma were necessary for the assay. For unconjugated estrogen, the blood level was quantitatively measured irrespective of applied plasma volume by 2 step extraction, since solid-phase extraction with liquid-liquid extraction eliminate water-and oil-soluble contaminants. By coupling solid-phase extraction and a specific RIA system, changes in peripheral estrone sulfate were determined during the regular estrous cycle and early stages of singleton and twin pregnancies caused by transfer of IVF embryos. Estrone sulfate during regular estrous cycle showed significant correlation with the concentrations of unconjugated estrogens. Peripheral estrone sulfate is not an appropriate indicator for follicular development in cattle because its concentration has a smaller range compared to estradiol-17β. In pregnant cows, no significant changes due to pregnancy were observed until Day 45 at any concentration of estrogen. Since Day 50, estrone sulfate concentration is elevated with the advance of pregnancy. Peripheral concentrations of unconjugated estrogens were kept at basal levels until Day 59. The timing of increment in estrone sulfate concentration in twin pregnant cows is earlier than in cows with singleton pregnancy. The rate of increase in estrone sulfate concentration was higher in twin carrying cows than singleton cows. Peripheral estrone sulfate would thus appear a useful indicator of placental formation in cattle because increase in estrone sulfate production is accompanied by development of the placenta. A reliable solid-phase extraction of estrogens from bovine blood was established. Changes in estrone sulfate concentration in bovine blood plasma during regular estrous cycle and early pregnancy were determined.

1998-03-25

乙第5327号

博士(獣医学)

博士論文

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