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Biomedical subjects

E Diczfalusy

Publications and source records attributed to E Diczfalusy.

At least 127 records · Page 7Linked to original sources
PubMed1979

Chemical synthesis and bioassay of anordrin and dinordrin I and II.

The chemical synthesis of 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-androstane-2 beta, 17 beta-diol dipropionate (Anordrin) and the corresponding diacetate is reported. Similarly, the preparation of the 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-estrane-2 beta, 17 beta-diol, its diacetate and dipropionate (Dinordrin I), along with the corresponding 2 beta-epimer (Dinordrin II) from 17 beta-hydroxy-A-nor-5 alpha=estran-2-one is described. In rat uterotrophic activity bioassay, the slope of ethynylestradiol differed significantly from the slopes of the other three compounds, thus vitiating potency estimates with this reference compound. Dinordrin I was 20 times more potent than Anordrin and considerably more potent then Dinordrin II. The single-dose oral antifertility effect in rats generally paralleled uterotrophic activity. Immediate postovulatory contraceptive effectiveness was assessed in adult cycling female baboons given two doses daily for 4 days. Both Anordrin and Dinordrin I showed antifertility activity worthy of further study. Moreover, a definite luteolytic effect, with depression of both plasma estrogen and progesterone levels, was observed with these two steroids.

Animals↗
PubMed1979

Effect of stress on the profile of plasma steroids in baboons (Papio hamadryas).

Adult baboons (5 males and 5 females) were exposed to immobilization stress by being strapped to a table in a horizontal position for 2 h. In females the experiment was performed during both the follicular and luteal phase. Peripheral blood was withdrawn at frequent intervals, the first sample just before immobilization, and the last one 3 days later. A number of steroids were measured in blood plasma samples by radioimmunoassay (17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnenolone, testosterone, dihydrotestosterone, progesterone, 20alpha-dihydroprogesterone, oestone, oestradiol) or competitive protein binding (cortisol) techniques. The cortisol levels exhibited a marked increase in both sexes. This increase was observed already during the immobilization and lasted for approximately 24 h. A similar, even more pronounced increase was seen in 17-hydroxypregnenolone, 17-hydroxyprogesterone and pregnenolone levels. A marked, long-lasting (72 h) decrease of testosterone and dihydrotestosterone levels was a consistent finding in male baboons. This was not observed in the females which, on the other hand, exhibited a marked decrease (duration 48 h) of progesterone and 20alpha-dihydroprogesterone levels during the luteal phase, and a significant decrease (duration greater than 24 h) of oestradiol and oestrone concentrations during the follicular phase. It is concluded that stress has a marked inhibitory action on gonadal function both in male and female baboons. In females and inhibition of steroidogenetic function is exerted both on the ovarian follicles and on the corpus luteum.

17-alpha-Hydroxypregnenolone↗
PubMed1979

A sensitive and specific in vitro bioassay method for the measurement of follicle-stimulating hormone activity.

An in vitro bioassay method for hFSH is presented. The method is based on the principles previously described by Dorrington et al. (1976b) and involves the assay of oestradiol produced from 19-hydroxyandrostenedione by dispersed Sertoli cells of 10-day old rats when cultured in the presence of graded doses of FSH. Using the 1st International Reference Preparation for human pituitary gonadotrophins (FSH and LH/ICSH) for bioassay (code no. 69/104) as standard, the useful range of the method is from 0.5 to 32 mIU/chamber (2 to 128 mIU/ml). The sensitivity of the method is 0.5 mIU/chamger. The mean index of precision (lambda) obtained from 16 multiple assays over 2 or 3 dose levels was 0.084. Parallelism was obtained between the 69/104 preparation and all preparations under study. The practicability of the proposed assay method is such that 15 preparations at 3 dose levels can be assayed by one person in 3 days. The specificity of the assay was investigated by determining the FSH activity in the following preparations: hFSH alpha- and beta-subunits, hLH, hCG, hTSH, ACTH, human growth hormone (hGH) human prolactin (hPRL) and luteinizing hormone-releasing hormone (LH-RH). The ACTH, hGH, hPRL and LH-RH preparations studied showed no detectable FSH activity in the assay. In the remaining preparations very low levels of FSH activity were found, corresponding to 0.004 to 0.6% of the weight of these preparations when compared with a highly purified hFSH preparation, suggesting that the method is specific for FSH. The possible synergistic or antagonistic influence of the above preparations when assayed in the presence of the 69/104 preparation was also assessed. No evidence of a synergistic or antogonistic effect was found. The assay of the hFSH potencies of a limited number of hFSH preparations of varying purity by the proposed in vitro bioassay, an hFSH radioreceptor method and an hFSH specific radioimmunoassay technique revealed that - although the relationship of the various potencies obtained with each method showed a close agreement - the bioassays yielded the highest potency estimates, and the radioimmunoassays the lowest ones. Since the proposed bioassay method is sensitive and considered to be specific for hFSH activity, it provides a suitable basis for the assessment of the specificity of other in vitro methods (radioreceptor and radioimmunoassay) currently used for detecting low levels of FSH activity.

Age Factors↗
PubMed1979

Influence of the purity of the iodinated tracer on the specificity of the radioimmunoassay of human follicle-stimulating hormone.

The objective of this study was to establish whether [125I]hFSH subunits are formed during iodination of hFsh by a mild (lactoperoxidase) technique, and if so which fractionation procedure provides the best separation of [125I]hFSH from its subunits. In addition, the effect of the 125I subunits on the specificity of the hFSH radioimmunoassay technique was examined. The products of iodination of hFSH were fractionated by a number of techniques consisting of low and high resolution gel filtration procedures and adsorption on either cellulose of Concanavalin A coupled to Sepharose. The efficacy of the adsorption methods was assessed by a subsequent high resolution gel filtration procedure (Ultrogel AcA 54). Radioreceptor and radioimmunoassay methods were used to monitor the profiles of hFSH, hFSH alpha and hFSH beta subunits and to assess the recoveries of radioreceptor-active hFSH obtained by the different procedures. Iodination of a highly purified hFSH preparation invariably resulted in the formation of both [125I]hFSH subunits. The proportion of radioactivity associated with the 125I subunit peak to that of the [125I]hFSH peak was 10% when the iodination products after low resolution gel filtration were fractionated by a high resolution gel filtration procedure. The corresponding amount of subunits obtained by adsorption on cellulose and Concanavalin A was 71% and 6%, respectively. Thus in comparison with low resolution gel filtration there was a partial separation of [125I]hFSH from [125I]hFSH subunits on Concanavalin A while no separation was effected on cellulose. In fact, the results suggest that 125I subunits were formed during fractionation on cellulose. The recoveries of hFSH activity following fractionation on cellulose and Concanavalin A were significantly lower (27% and 65%) than those obtained by either low or high resolution gel filtration techniques. A marked reduction ;n the specificity of the RIA of hFSH was found when [125I]hFSH subunits rather than [125I]hFSH were used as tracer in the assay. It is concluded that the presence of 125I subunits in [125I]hFSH preparations used as tracer in the RIA of hFSH can diminish the specificity of the assay. The routine employment of a high resolution gel filtration procedure which provides a complete separation of 125I subunits from the intact hormone, is suitable for the elimination of this source of assay invalidity while the other fractionation procedures tested (cellulose adsorption or Concanavalin A) are not.

Adsorption↗
PubMed1979

Biologically active luteinizing hormone (LH) in plasma. III. Validation of the in vitro bioassay when applied to male plasma and the possible role of steroidal precursors.

An in vitro bioassay method for measuring LH activity was applied to male plasma. This method is based on the specific testosterone response to LH activity by interstitial cells from mouse testes. In contrast to assays conducted on female plasma, non-parallel response lines were obtained between serial dilutions of untreated male plasma and the International Reference Preparation for Human Pituitary Gonadotrophins FSH and LH/ICSH) for bioassay (code no. 69/104). In an attempt to eliminate this source of error, which would invalidate the assays, plasma was subjected to either ether extraction or charcoal adsorption prior to assay. While ether extraction was ineffective, charcoal treatment eliminated the source of non-parallelism. Evidence is presented indicating that the inclusion of a charcoal pre-treatment step provides an assay method for LH which fulfils the recognized criteria of reliability when applied to male plasma. An investigation of the likely causes of non-parallelism was undertaken by incubating mouse interstitial cells with various steroids and steroid sulphates at concentrations likely to be present in plasma. While most of the presumed precursors of testosterone were converted to testosterone, steroid sulphates (dehydroepiandrosterone sulphate and pregnenolone sulphate) at high concentrations as present in male plasma were the most active compounds in forming testosterone. However, the amount of testosterone produced from these precursors under controlled conditions was insufficient to account entirely for the deviation from parallelism observed with male plasma. Hence, the non-parallelism observed with untreated plasma samples cannot be entirely explained by the presence of steroidal testosterone precursors in male plasma.

Adsorption↗
PubMed1979

Biologically active luteinizing hormone (LH) in plasma. V. A re-analysis of the differences in the ratio of biological to immunological LH activities during the menstrual cycle.

The relationship between the biological and immunological activities of human luteinizing hormone (hLH) in plasma collected from female subjects was examined. The biological activity was measured by an in vitro bioassay and the immunological activity by an hLH radioimmunoassay (RIA), using improved reagents, such as the 1st IRP for human pituitary LH for immunoassay (code No. 68/40) as standard, a subunit-free biologically active iodinated hLH preparation as tracer and an anti-hLH serum of relatively high specificity. Similar profiles of biological (B) and immunological (I) activity were obtained in the plasma samples collected daily throughout 40 menstrual cycles (5 cycles from each of 8 subjects). However, the B/I ratios were significantly lower during the period of LH surge (P less than 0.001) than throughout the remainder of the cycle. The within- and between-assay variation in B/I ratios was investigated by the simultaneous assay of biological and immunological activities in plasma pools obtained by combining equal aliquots of plasma from each daily sample of the menstrual cycle from each of 5 cycles of each of 4 subjects. The analysis of these 20 pools revealed highly significant individual differences in B/I ratios, ranging from 0.81 to 1.33. The coefficient of variation was 20% between-subjects and 5% within-subjects. There was no seasonal variation in B/I ratios. That the individual differences in plasma B/I ratios were not attributable to the procedure of pooling was ascertained by the simultaneous assay of both activities in parellel in daily plasma samples and in the pools formed from these samples from three complete cycles. Thus the analysis of the differences in B/I ratios obtained throughout the menstrua- cycle revealed three major sources of variation. The first occurs in the form of generally elevated (higher than unity) B/I ratios, the second consists of a significant drop in B/I ratios during the midcycle LH surge, and the third source is represented by the significant between-subject differences. It is concluded that the first source is attributable to the relatively higher levels of "impurity" (i.e. biologically inactive, immunologically active material) in the standard preparation compared to those present in plasma of biologically inactive, immunologically active material of unknown composition and origin. If so, the latter source limits the quantitative significance of the RIA procedures employed. It is suggested that these three sources of variation account for most of the differences in B/I ratios for plasma lLH reported in the literature.

Adult↗
PubMed1978

Biological and immunological characterization of human luteinizing hormone: IV. Biological and immunological profile of two international reference preparations after electrofocusing.

The first IRP of Human Pituitary Gonadotrophins (FSH/LH) for bioassay (69/104) and the 1st IRP of Human Pituitary Luteinizing Hormone (LH) for immunoassay (68/40) were fractionated by an electrofocusing technique in a sucrose density gradient and the profile of biological and immunological activities was determined. The partially purified LH preparation (69/104) gave a broad pattern of biolgical and immunological activities which extended from pH 4 to 10. The biological : immunological (B/I) ratio of the various fractions (using the 68/40 preparation as standard) ranged from 0.04 to 1.05. The low B/I ratios indicate the presence of high levels of immunologically active, biologically inactive material in this preparation. In contrast to the behaviour of the 69/104 preparation, the major proportion (88%) of the biological activity recovered after electrofocusing of the highly purified LH preparation (68/40) was found within the pH range 7--9, with B/I ratios (again using as standard the 68/40 preparation) ranging from 0.4 to 1.5. Multiple dose parallel line design radioimmunoassays revealed a lack of parallelism between the 69/104 and 68/40 International Reference Preparations. This was attributed to the presence of acidic material in the former preparation, which is practically absent from the latter. The biological LH profiles of both the 69/104 and 68/40 preparations differed from those previously reported for aqueous extracts of pituitaries. It is concluded that the dissimilarity in the electrofocusing profiles of the biological and immunological activities of the 69/104 preparation renders this preparation unsuitable as a reference preparation for the quantitation of biologically active LH by radioimmunoassay methods. Using the same criteria, the 68/40 preparation would appear to be a more suitable standard.

Animals↗
PubMed1978

Effect of graded oral doses of oestradiol on circulating hormonal levels.

A single oral dose of micronized oestradiol (1, 2 or 4 mg) was administered to 10 normally menstruating women on the second day of 3 consecutive menstrual cycles. The order in which the 3 doses were given was chosen randomly for each subject. Samples of peripheral blood were withdrawn on 3 occasions (10, 5 and 1 min) before and on 9 occasions (0.37, 0.75, 1.5, 30, 6.0, 12.0, 24.0, 48.0 and 72.0 h) after the ingestion of oestradiol. Oestradiol, oesterone, oestradiol sulphate, oestrone sulphate, testosterone and LH were measured by means of radioimmunoassays in blood plasma on all occasions. The levels of all oestrogens increased significantly following the ingestion of oestradiol, although to a greatly different extent. The largest and most prolonged increase was seen in the case of oestrone sulphate, the levels of which--depending on the dose ingested--were 20-50 times higher than the pre-treatment levels 3 after the ingestion of the oestradiol capsule. The increase of the levels of oestrone sulphate was still significant (P less than 0.001) after 24 h, and following the ingestion of 2 and 4 mg of oestradiol even after 72 h (P less than 0.05). The levels of oestrone and oestradiol sulphate exhibited smaller increases 3 h after ingestion, i.e. 4-10 and 2.5-5 times, respectively, and the plasma levels of oestradiol showed a minor increase only (1.4-1.8 times). The concentration of oestradiol metabolites studied increased proportionally with the dose of oestradiol administered. On the other hand, a clear-cut dose--effect relationship could not be observed between the amount of oestradiol administered and the plasma levels of LH and testosterone. The data indicate that the bulk of perorally administered oestradiol is transformed to oestrone sulphate accompanied by smaller quantities of oestrone and oestradiol sulphate and that the ratios of circulating oestrogens seen after the ingestion of oestradiol deviate considerably from those observed in the normal menstrual cycle.

Administration, Oral↗
PubMed1978

Studies on a human chorionic gonadotrophin-like material present in non-pregnant subjects.

The presence of an hCG-like material in urinary and pituitary extracts and plasma obtained from non-pregnant subjects was investigated. Two assay methods were used to detect this material following fractionation of pituitary and urinary extracts by gel filtration (Ultrogel AcA 54) and/or isoelectrofocusing: a) a radioimmunoassay employing an antiserum raised against a specific sequence of the carboxy terminal region (residues 115--145) of the beta-subunit of hCG, and b) an in vitro bioassay method which measures both hLH and hCG activities. The fractionation procedures employed provide a satisfactory separation of highly purified hCG and hLH preparations. In the pituitary and urinary extracts hCG beta-peptide-like immunoactive (PIA) material was found consistently, which co-eluted with iodinated hCG following gel filtration and possessed pI values similar to those of hCG when subjected to isoelectrofocusing. The PIA material also exhibited in vitro biological activity similar to that shown by hLH and hCG. Detectable levels of immunoactive material were also found in plasma; however, the plasma levels of this PIA material were not influenced by classical endocrine measures such as the stimulation or inhibition of gonadotrophin secretion. The low levels of this material in plasma precluded its further characterization by gel filtration or electrofocusing. Whereas the present data and those reported by other investigators seem to suggest the presence of some hCG-like material in urinary and pituitary extracts and possibly in plasma of non-pregnant subjects, it is emphasized that the available evidence is not sufficiently conclusive to exclude other interpretations as to the nature of this material.

Biological Assay↗
PubMed1978

Influence of the purity of the iodinated tracer on the specificity of the radioimmunoassay of human luteinizing hormone.

Human luteinizing hormone (HLH) iodinated with 125I by the use of a lactoperoxidase method, was fractionated by either cellulose adsorption, gel filtration or by the combination of these methods. The products of iodination were characterized by their in vitro biological LH activity and by their binding profiles with antisera to HLH, HLHalpha and HLHbeta subunits. Several radioactive components were obtained after gel filtration with or without an initial cellulose adsorption step. One of these fractions was identified as biologically active HLH and another as the HLHalpha subunit. Radioimmunoassay studies were conducted with different iodinated fractions as tracers, using three well defined and widely available antisera to HLH. The standard used was the HLH International Reference Preparation for immunoassay (68/40). Cross-reactivity was examined with several purified pituitary preparations, such as HFSH, HTSH, HLHalpha and HLHbeta subunit. A significantly higher cross-reactivity with HLHalpha, HFSH and HTSH was obtained with the [125I]HLHalpha fraction as tracer than with biologically active [125I]HLH. Furthermore, in the radioimmunoassay of HLH preparations of varying purity, significantly higher estimates of immunological activity were obtained with the [125I]HLHalpha tracer than with the biologically active [125I]HLH. It is concluded that the presence of [125I]HLHalpha in the [125I]HLH tracer can result in serious overestimates of the immunological activity in the measurement of LH. Therefore [125I]HLHalpha should be separated from [125I]HLH prior to radioimmunoassay. Many of the fractionation methods commonly used (cellulose adsorption and short column gel filtration systems) are inadequate for this purpose. However, an adequate separation can be achieved by the use of high resolution gel filtration systems.

Biological Assay↗
PubMed1977

Biological and immunological characterization of human luteinizing hormone: I. Biological profile in pituitary and plasma samples after electrofocusing.

Pituitary and plasma pools from postmenopausal women and plasma pools from women at midcycle were fractionated by electrofocusing in sucrose density gradients. The biological LH activity was determined in each of the electrofocusing fractions by the use of an in vitro bioassay method. A heterogeneous profile of LH activity was found in both pituitary and plasma samples with a large proportion present within the pH range 6.5-10. In a total of 11 electrofocusing runs 7 main regions of high LH activity were found within this range with mean pI values (+/- SD) of 6.75 +/- 0.08 (n = 6), 7.33 +/- 0.08 (11), 7.80 +/- 0.09 (11), 8.23 +/- 0.10 (11), 8.81 +/- 0.04 (7), 9.17 +/- 0.05 (6) and 9.55 (2). A significantly higher proportion of LH activity was found in the midcycle plasma samples (36%) in the pH regions with mean pI values of 8.81, 9.17 and 9.55 than in postmenopausal plasma (7%) and pituitary extracts (5%). This indicates that the profile of biologically active LH in women in the fertile age is different from that present in postmenopausal women. By detailed fractionation based on narrow pH range studies and the refocusing of specific peak fractions it was shown that each of the regions studied consisted of several peaks of LH activity indicating the presence of a large number of molecular species exhibiting varying degrees of LH activity. The relative proportions of these species showed considerable differences between sources but also between samples from the same source.

Animals↗