A radioimmunoassay method for simultaneous determination of pregnenolone, pregnenolone sulfate, dehydroepiandrosterone and dehydroepiandrosterone sulfate in human plasma (Japanese)

Abstract

The method consists of the following procedures: 1) ether extraction of unconjugated compounds, 2) extraction of sulfates from aqueous residue with ethyl acetate, 3) solvolysis with sulfuric acid at 40°C for 60 minutes, 4) celite column chromatography to separate individual compounds, 5) radioimmunoassay. The efficiency of solvolysis for pregnenolone sulfate and DHA sulfate is 94 and 80%, respectively. Precision and accuracy studies have shown that the assays of sulfates as well as unconjugates are reproducible and accurate. Specificity was ascertained by parallelism and linearity studies. No interfering substance was detected in an appreciable quantity. Plasma levels of these four compounds were determined in specimens obtained from 15 normally ovulating women. To represent the whole menstrual cycle, samples were taken 8 days before LH peak (LH-8), the day of LH peak (LH=0) and 8 days after LH peak (LH+8). Plasma contents of these compounds (geometric mean in ng/ml and 95% confidence limits in parentheses) are as follows: pregnenolone, LH-8: 1.33 (1.02-1.74), LH=0: 1.45 (1.22-1.72), LH+8: 1.88 (1.70-2.21); pregnenolone sulfate, LH-8: 70.0 (55.9-89.2), LH=0: 57.5 (40.0-82.7), LH+8: 102 (81.15-12.9); DHA, LH-8: 5.38 (3.90-7.43), LH=0: 4.90 (3.58-6.79), LH+8: 4.58 (3.12-6.83); DHA sulfate, LH-8: 1480 (1110-1980), LH=0: 1570 (1150-2140), LH+8: 1590 (1150-2190). Both pregnenolone and pregnenolone sulfate levels of 8 days after LH peak are significantly higher than those of the other two days. Conversely, plasma DHA and DHA sulfate levels fluctuate over a wide range with no consistent trend.