血中17α-hydroxypregnenolone, 17α-hydroxypregnenolone sulfateおよび17α-hydroxyprogesteroneの同時測定法

抄録

It is well recognized that in the fetal adrenal cortex, 3β-hydroxysteroid dehydrogenase (3β-HSD) activity is lower and in fetal tissues, steroid sulfokinase (SK) is higher than in the adult. In order to clarify a part of the developmental processes of the adrenal cortex or steroidgenesis in humans, a combined radioimmunoassay (RIA) method to estimate serum 17α-hydroxypregnenolone (17-OH-Δ⁵P), 17α-hydroxypregnenolone sulfate (17-OH-Δ⁵ P-S) and 17α-hydroxyprogesterone (17-OH-Δ⁴P) was devised.
The method consisted of the following procedures : 1) diethyl ether extraction and chromatographic separation of unconjugated steroids (17-OH-Δ⁵P and 17-OH-Δ⁴ P), 2) enzymatic hydrolysis of 17-OH-z5P-S using the residue of diethyl ether extraction for a material, 3) diethyl ether extraction and chromatographic purification of hydrolyzed 17-OH-Δ⁵P-S, and 4) RIAs for 17-OH-Δ⁵ -Pto estimate 17-OH-Δ⁵P and 17-OH-Δ⁵P-S concentration, and for 17-OH-Δ⁴P.
Extracted 17-OH-Δ⁵P was well separated from 17-OH-OΔ⁴P by Sephadex LH-20 microcolumn chromatography, using a benzen/methanol= 95/5 (v/v) solvent as a mobile phase.
Several procedures for hydrolysis or solvolysis of 17-OH-Δ⁵P-S were compared using available tritiated Δ⁵-3β-hydroxysteroids including dehydroepiandrosterone (DHA), DHA sulfate (DHA-S) and 17-OH-Δ⁵5P, and it was found that the most suitable method was an enzymatic hydrolysis by arylsulfatase from Helix Pomatia in an appropriate condition in which the percent hydrolysis was 92.9 ± 1.2 (mean ± SEM)%.
The final percent recoveries were 88.7 ± 1.2% in 17-OH-Δ⁴ P, 90.7 ± 1.4% in 17-OH-Δ⁵P and 78.1 ± 2.1% in 17-OH-Δ⁵P-S, respectively.
A suitable antiserum and its final dilution titer for RIA of 17-OH-Δ⁵P (hydrolyzed 17-OH-Δ⁵P-S also) was 1:12,000 dilution of anti-17-OH-Δ⁵P-3-succinate-BSA serum. An anti-7-oxo-17-OH-Δ⁵P-7-carboxymethyloxime-BSA serum was considered to be unsuitable for the measurement of hydrolyzed 17-OH-Δ⁵5P-S, presumably because of a significant cross-reactivity with a large amount of unknown steroid sulfates simultaneously hydrolyzed.
The fact that concentrations of the steroids estimated by this method were well in agreement with those previously reported by other authors and that the precision and accuracy of this method were both satisfactory indicates the availability of this combined RIA method for the elucidation of developmental changes in the activities of 3β-HSD and SK in infancy and childhood.