Simultaneous determination of the sulphates of dehydroepiandrosterone and pregnenolone in plasma by radioimmunoassay following a rapid solvolysis.
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Biomedical subjects
Publications and source records attributed to T Aso.
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The plasma levels of estradiol and progesterone in 5 mature female Japanese monkeys (Macaca fuscata fuscata) throughout a breeding and nonbreeding season were measured by radioimmunoassay. As the breeding season approached, plasma estradiol levels started to increase and a few elevations were detected. However, neither a plasma progesterone rise nor menstrual bleeding was seen. Menstrual bleeding began in December and recurred regularly 4 or 5 times at 4 week intervals until April. During this period, cyclic estradiol elevations (150-250 pg/ml) and luteal progesterone rises (2.0-5.3 ng/ml) were observed. After the last menstruation in March or April, two animals showed an estradiol elevation, but progesterone levels remained in the basal range and amenorrhea persisted. These data indicated that female Japanese monkeys ovulate 4 or 5 times a year, i.e., their breeding season is definitely defined. The changes of both steroids in transitional periods between nonbreeding and breeding, and breeding and nonbreeding seasons, suggested that some follicle development occurred in these periods. The elucidation of the mechanisms involved in the seasonal variation of reproductive functions in the Japanese monkey may provide useful information concerning some aspects of amenorrhea in human females.
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A radioimmunoassay method has been developed for the simultaneous determination of pregnenolone, pregnenolone sulfate, dehydroepiandrosterone(DHA) and dehydroepiandrosterone sulfate (DHA sulfate). The method consists of the following procedures: 1) ether extraction of unconjugated compounds, 2) extraction of sulfates fromaqueous residue with ethyl acetate, 3) solvolysis with sulfuric acid at 40 degree C for 60 minutes, 4) celite column chromatography to separate individual compounds, 5) radioimmunoassay. Efficiencies of solvolysis for pregnenolone sulfate and DHA sulfate are 94 and 80%, 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 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 = O) and 8 days after LG 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 (81.5-129); 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 other two days. Conversely, plasma DHA and DHA sulfate levels fluctuate over wide range with no consistent trend.
Eleven unconjugated steroids were measured daily during a complete cycle in the peripheral plasma of 6 normally menstruating baboons (Papio hamadryas) by means of a radioimmunoassay procedure and the levels were compared with those found previously in 17 normally menstruating women. The patterns of progesterone and 20alpha-dihydroprogesterone were very similar to those found in women throughout the entire menstrual cycle. However, the ratio of these steroids differed markedly from that found in women. A great similarity of the follicular phase and the peri-ovulatory period profiles was observed in both species for 17-hydroxy-progesterone, pregnenolone, androstenedione, oestradiol and oestrone. The pattern of oestradiol:oestrone ratios was similar in both species. However, an elevation of the above five steroids, typical for the human luteal phase, was not found in baboons. The increase of testosterone values, seen in women at mid-cycle, was not detected in baboons. The plasma concentrations were lower in baboons than in humans for all the above steroids.
In an attempt to find suitable animal models to aid in the study of the reproductive processes of the human male, plasma levels of unconjugated pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, 20alpha-dihydroprogesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone, oestrone and oestradiol were measured in 18 male baboons and 10 male rhesus monkeys and the steroid levels were compared with those previously established in normospermic, middle-aged men. Significant species differences were found with regard to the three delta5-steroids studied; whereas the approximate relationship of pregnenolone to 17-hydroxypregnenolone and dehydroepiandrosterone was 1:2:4 in men, the corresponding relationship was 1:5:30 in rhesus monkeys and 1:10:10 in baboons. Similar levels of 20alpha-dihydroprogesterone were found in the three species. On the other hand, the 17-hydroxyprogesterone levels in baboons were much lower and the levels of androstenedione lower than those found in men and in rhesus monkeys. No species difference was found with regard to circulating testosterone levels. However, both rhesus monkeys and baboons exhibited much higher levels of dihydrostestosterone than did men. Oestrone levels were higher in baboons than in men and oestradiol levels were higher in rhesus monkeys than in men and in baboons. The significant differences in circulating steroid levels suggest that further studies (including i.a. steroid analyses in testicular tissue, seminal plasma and spermatic artery and vein following both stimulation and suppression of testicular endocrine function) are required before preference can be given to any of the two species studied as a suitable animal model for the study of new fertility regulating agents.
In an attempt to analyze the multiple changes and interactions in circulating steroid levels in the peri-ovulatory and peri-menstrual periods, the plasma levels of immunoreactive luteinizing hormone (LH), progesterone and unconjugated pregnenolone, dehydroepiandrosterone, testosterone, oestradiol and oestrone were assayed daily during a complete cycle in 17 normally menstruating women. In 14 of the 17 subjects studied androstenedione and unconjugated dihydrotestosterone were also estimated. The day of the LH-peak and the first day of menstruation, respectively, were used to synchronize the peri-ovulatory and peri-menstrual plasma levels of the various steroids. With the exception of dehydroepiandrosterone and dihydrotestosterone, the plasma levels of all steroids exhibited significant, but different changes during the cycle. Testosterone levels showed a slight but significant increase around the LH-peak, whereas the levels of pregnenolone and androstenedione were higher in the post-ovulatory than in the pre-ovulation periods. The levels of oestradiol and oestrone, as well as the ratios of oestradiol to oestrone gradually increased from the low values observed in the early proliferative phase to pre-ovulatory peak values. The relationship between peaks of oestradiol and oestrone and that of LH exhibited great individual variation. The same was true for the individual oestradiol to oestrone ratios. The combination of several steroidal signals did not improve the predictive value of the analyses. However, an increase of individual progesterone values by at least 0.35 ng/ml from the day preceding the LH-peak to the day of the LH-peak was observed in 13 of the 17 subjects. It is suggested that for the early detection of the LH surge and prediction of the subsequent ovulation daily assays of plasma progesterone are of more value than the assay of the other steroids investigated.
A rapid 5 h radioimmunoassay method for the determination of progesterone and oestradiol in the plasma of non-pregnant women is described. Due to the high specificity of the antisera used, it is possible to perform the radioimmunoassay directly on the ether extracts of plasma, without employing chromatographic purification of the steroids. Evidence is presented indicating that the rapid assay is almost as reliable as the previously described radioimmunoassay method which involves chromatography. The within-assay and between-assay coefficients of variation in the progesterone assay were 7-74 and 14-9 and in the oestradiol assay 7-36 and 18-1 respectively. Comparisons between increasing doses of authentic hormone and endogenous hormone extracted from plasma indicated no deviation from parallelism. Progesterone and oestradiol were assayed in 300 plasma samples by the rapid method and by the method involving chromatography. The slopes obtained by a regression analysis were close to unity for both progesterone and oestradiol (1-04 and 1-06, respectively), the y-intercepts were - 0-21 and 0-16 and the correlation coefficients 0-98 and 0-88, respectively. When the data obtained by both techniques in fourteen menstrual cycles were compared, the results were practically identical. In ten repeated studies conducted by four investigators it was shown that two workers can complete the assay of both progesterone and oestradiol in twenty-five plasma samples in duplicates with 5 h. The same time required for the assay of either progesterone or oestradiol in twenty-five duplicates by one worker.
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To examine androgenic potential of polycystic ovaries (PCO), slices of follicular and stromal tissues from the same ovary obtained by wedge resection from two markedly, two moderately and one slightly enlarged PCO were incubated separately with [1-14C] acetate. Incorporation into progestins, androgens and estrogens was assessed by reverse dilution technique with recrystallization to constant specific activity. Although the greatest incorporation into androstenedione with much lesser incorporation into testosterone and dehydroepiandrosterone was observed with all the five follicles, the amount of incorporation into the three steroids increased gradedly with histologically defined magnitude of thecal cell hyperplasia in atretic follicles. Only the stromal tissues from two markedly enlarged PCO produced androgens with a similar pattern of 14C distribution among the steroids, thereby incorporation into the three androgens remaining 5.5% or less of that by the follicles from the same ovary. Preoperative levels of plasma androstenedione and urinary 17-ketosteroids were shown to increase in four patients with PCO containing atretic follicles with thecal cell hyperplasia, but not in one patient with slightly enlarged PCO containing atretic follicles without thecal cell hyperplasia. It is inferred that atretic follicles with thecal cell hyperplasia is a significant source of androgen overproduction by PCO.
The objective of this article is to investigate the clinical features of pulmonary metastasis (PM) from endometrial adenocarcinoma, in particular, the predictors of prolonged survival after PM detection. Fifteen patients who developed PM and underwent chest computed tomography (CT) scans for evaluation of PM were studied: 12 patients with pulmonary recurrence and 3 patients with PM on presentation. All patients with bilateral nodules or lymphangitic spread had metastases in other sites prior to or concomitant with PM, most of which were detected in para-aortic lymph nodes and/or the vaginal wall, while only one of five patients with a limited number (n < or = 5) of unilateral nodules had these metastases. The median survival time for the 10 patients with bilateral PMs was significantly shorter than that of the 5 patients with a limited number of unilateral nodules who were treated with surgery (7 versus 50 months, P = 0.005). Patients who developed pulmonary recurrence 2 years after the initial therapy had a significantly longer survival than those who developed it within 2 years (31 versus 10 months, P = 0.01). In conclusion, the distribution of PM determined by CT scans and the time interval between the initial therapy and the detection of pulmonary recurrence are the predictors of survival after PM detection.
This is the first attempt to elucidate the direct effect of acupuncture stimulation on plasma levels of LH, FSH, progesterone and esterone and estradiol in normally ovulating women. Four loci, which are known from experience to relate closely to female reproductive organs, were needled with electric stimulation and the responses to synthetic LH-RH with and without acupuncture stimulation were also analyzed in the same cases. Plasma hormone levels were measured by specific radioimmunoassay. All cases showed normal responses to LH-RH corresponding to the day of the menstrual cycle. During the initial part of stimulation, both progesterone and estradiol levels rose in follicular cases and declined in luteal cases. Plasma LH levels declined and FSH showed no change at this time. The analysis of plasma LH responses to LH-RH under acupuncture stimulation revealed extraordinary re-elevation in some cases. It is suggested that properly performed acupuncture stimulation might affect the female endocrine function.