Metabolism of estrogens and androgens by scleractinian corals.

Estrogens and androgens are steroids that act as reproductive hormones in vertebrates. These compounds have also been detected in reef-building corals and other invertebrates, where they are hypothesized to act as bioregulatory molecules. Experiments were conducted using labeled steroid substrates to evaluate metabolism of estrogens and androgens by coral homogenates. GC-MS analysis of 13C-labeled steroids showed that Montipora capitata coral homogenates or fragments could convert estradiol to estrone and testosterone to androstenedione and androstanedione, evidence that M. capitata contains 17beta-hydroxysteroid dehydrogenase and 5alpha-reductase. When homogenates from three coral species and symbiotic dinoflagellates (zooxanthellae) were incubated with tritiated steroid substrates, metabolites separated by thin-layer chromatography confirmed that 17beta-hydroxysteroid dehydrogenase activity occurred in all species tested. NADP+ was the preferred cofactor in dehydrogenation reactions with coral homogenates. Reduction of estrone and androstenedione occurred at lower rates and aromatization of androgens was not observed. It is unclear whether estrogens detected previously in coral tissues are produced endogenously or sequestered in coral tissue from dietary or environmental sources. Previous studies have demonstrated that corals can take up estrogens from the water column overlying coral reefs. Considered in total, these observations suggest corals could alter the concentration or form of steroids available to reef organisms.

Androgen formation and metabolism in the pulmonary epithelial cell line A549: expression of 17beta-hydroxysteroid dehydrogenase type 5 and 3alpha-hydroxysteroid dehydrogenase type 3.

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A549, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A549 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A549 cells. When cultured in the presence of normal FCS, A549 intact cells converted DHEA to androstenediol, androstenedione principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A549 RNA indicated the presence of 17beta-HSD type 4 and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, androstenedione was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A549 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

Tissue- and site-specific gene expression of type 2 17beta-hydroxysteroid dehydrogenase: in situ hybridization and specificenzymatic activity studies in human placental endothelial cells of the arterial system.

Progesterone and estradiol are the most potent human sex steroid hormones of placental origin and are essential to the maintenance of pregnancy, the timing of parturition, the maturation of many fetal organs, and the preparation of the maternal reproductive system. Naturally, regulatory mechanisms must be in place to coordinate the synthesis and inactivation of these two hormones. We have previously shown that the highest levels of type 1 and type 2 17beta-hydroxysteroid dehydrogenase (17betaHSD) messenger ribonucleic acids (mRNAs) occur in the placenta, particularly in the villi. However, in contrast to type 1 17betaHSD mRNA, type 2 17betaHSD mRNA was not detectable in cell cultures of human cytotrophoblasts or syncytiotrophoblasts. Using in situ hybridization, we unequivocally identified endothelial cells as the only cell type expressing the type 2 17betaHSD gene in fetal villi. Moreover, type 2 17betaHSD mRNA was specifically detected in the endothelial cells of the arterial system, and at higher levels in the villi compared with endothelial cells of the cord arteries when the two tissue sections were cohybridized. In fact, both mRNA levels and enzymatic activity are at their highest levels in arterial endothelial cells. In conclusion, the endothelial cells of the villous arterioles are the primary site of type 2 17betaHSD gene expression. This suggests a regulatory role for these cells in the control of progestin, androgen, and estrogen levels during pregnancy, thus opening a whole new way of viewing regionalization and localization of steroidogenesis in the human villi.

Separation by thin-layer chromatography of the most common androgen-derived C19 steroids formed by mammalian cells.

Several methods have been developed in the past for the separation and identification of closely related steroid hormones. Although these methods are effective, most of them use HPLC-derived systems and are expensive, laborious, or time-consuming. In the course of our studies of the metabolism of dehydroepiandrosterone and androstenedione in tissues, we have modified a previously published technique in such a way that in one TLC step we can separate most of the androgen C19 steroid derivatives produced by mammalian cells. We have used this modified technique for the past 2 years with considerable success and reproducible results, and we find it to be rapid and relatively inexpensive.

Expression of different 17beta-hydroxysteroid dehydrogenase types and their activities in human prostate cancer cells.

The 17beta-hydroxysteroid dehydrogenase (17betaHSD) enzyme system governs important redox reactions at the C17 position of steroid hormones. Different 17betaHSD types (no. 1-4) have been identified to date in peripheral human tissues, such as placenta, testis, and breast. However, there is little information on their expression and activity in either normal or malignant prostate. In the present work, we have inspected pathways of 17beta-oxidation of either androgen or estrogen in human prostate cancer cells (LNCaP, DU145, and PC3) in relation to the expression of messenger RNAs (mRNAs) for 17betaHSD types 1-4. These cell systems feature distinct steroid receptor status and response to hormones. We report here that high expression levels of 17betaHSD4 were consistently observed in all three cell lines, whereas even greater amounts of 17betaHSD2 mRNA were detected solely in PC3 cells. Neither 17betaHSD1 nor 17betaHSD3 mRNAs could be detected in any cell line. From a metabolic standpoint, intact cell analysis showed a much lower extent of 17beta-oxidation of both androgen [testosterone (T)] and estrogen [estradiol (E2)] in LNCaP and DU145 cells compared to PC3 cells, where a greater precursor degradation and higher formation rates of oxidized derivatives (respectively, androstenedione and estrone) were observed. Using subcellular fractionation, we have been able to differentiate among 17betaHSD types 1-4 on the basis of their distinct substrate specificities and subcellular localization. This latter approach gave rise to equivalent results. PC3 cells, in fact, displayed a high level of microsomal activity with a low E2/T activity ratio and approximately equal apparent Km values for E2 and T, suggesting the presence of 17betaHSD2. Dehydrogenase specific activity with both E2 and T was also detected, although at lower levels, in LNCaP and DU145 cells. No evidence for reductase activity could be obtained in either the soluble or microsomal fraction of any cell line. As comparable expression levels of 17betaHSD4 were seen in the three cell lines, 17betaHSD2 is a likely candidate to account for the predominant oxidative activity in PC3 cells, whereas 17betaHSD4 may account for the lower extent of E2 oxidation seen in both LNCaP and DU145 cells. This is the first report on the expression of four different 17betaHSD types in human prostate cancer cells. It ought to be emphasized that for the first time, analysis of different 17betaHSD activities in either intact or fractionated cells harmonizes with the expression of relevant mRNAs species.

Expression of the genes for 3 beta-hydroxysteroid dehydrogenase type 1 and cytochrome P450scc during syncytium formation by human placental cytotrophoblast cells in culture and the regulation by progesterone and estradiol.

We have investigated the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 1 genes during human trophoblast differentiation in culture and the modulation of their steady-state mRNA levels by steroids. During the first 24 to 48 h after plating, mononucleated cells aggregated, forming colonies. After 60 h in culture, cell diameters were increased and nuclei appeared centrally distributed within large cells, consistent with syncytiotrophoblast formation. During these striking morphological changes in culture the expression and activity levels of 3 beta-HSD type 1 and P450scc increased significantly as isolated cytotrophoblasts progressed to a differentiated state, with P450scc and 3 beta-HSD type 1 mRNAs activities being more abundant in cells cultured for 48 to 72 h. In the same culture, however, the amount of 3 beta-HSD protein decreased during the first 12 to 24 h by 50% compared with freshly isolated trophoblasts but remained at these levels throughout the culture period. The specific activity of the 3 beta-HSD as determined with pregnenolone or dehydroepiandrosterone was similar but increased with time as syncytiotrophoblast was formed in vitro. These observations provide additional evidence that the expression of these two progesterone-synthesizing enzymes is coincident and that they reach their maximum steady-state mRNA levels at a time when syncytium formation occurs in vitro. Incubation of trophoblast cells with progesterone or estradiol increased the abundance of P450scc and 3 beta-HSD type 1 mRNAs but had no significant effect on the amount of 3 beta-HSD protein. These observations of the regulation of 3 beta-HSD type 1 mRNA levels by steroids suggest a complex relationship of the mechanisms regulating transcription/mRNA processing and transduction of the 3 beta-HSD type 1 gene.

Intracellular regulation of 17 beta-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells.

There is growing evidence that various isoforms of 17 beta-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17 beta-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme-NAD or an enzyme-NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the delta 4 or delta 5 pathway is limited.

Properties and regulation of 17 beta-hydroxysteroid oxidoreductase of OVCAR-3, CAOV-3, and A431 cells: effects of epidermal growth factor, estradiol, and progesterone.

Although there is a growing body of evidence that 17 beta-hydroxysteroid oxidoreductase plays a role in the regulation of steroid levels in epithelial tumors of the endometrium and breast, our knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17 beta-hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR-3, CAOV-3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17 beta-hydroxysteroid oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basis of estradiol/testosterone activity ratios, all three cell lines appear to have type 2-like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGF, alone or in combination, were without effect on the enzymatic activity of OVCAR-3 cells. EGF decreased the activity of CAOV-3 cells slightly. In contrast, EGF stimulated A431 17 beta-hydroxysteroid oxidoreductase activity 7-8-fold over a 5-day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGF. With EGF, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGF alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGF to low-affinity receptors on A431 cells.

Kinetic analysis of enzymic activities: prediction of multiple forms of 17 beta-hydroxysteroid dehydrogenase.

An overview of the application of kinetic methods to the delineation of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17 beta-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17 beta-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17 beta-HSD type 1 is a cytosolic enzyme highly specific for C18 steroids such as 17 beta-estradiol (E2) and estrone (E1). 17 beta-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E2 and E1. Useful parameters for the detection of multiple forms of 17 beta-HSD appear to be the E2/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17 beta-HSD type 2. The 17 beta-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17 beta-HSD which differ from 17 beta-HSD type 1 and type 2 in their kinetic properties.

Gestational development of human placental 17 beta-hydroxysteroid oxidoreductase types 1 and 2.

Human placenta is a rich source of 17 beta-hydroxysteroid oxidoreductase (17-HOR) type 1, a cytosolic enzyme highly specific for 17 beta-oestradiol, and type 2, a microsomal form reactive with both oestradiol and testosterone. Although a number of studies have established that 17-HOR activity is present in placenta as early as weeks 4-5 of gestation, more specific data on the pattern of development of these two enzyme forms are lacking. In this study, samples of villous tissue from weeks 7-20 of gestation were fractionated into cytosol and microsomes and 17-HOR activity assayed under conditions which differentiate between the two enzyme types. Type 1 activity with oestradiol of cytosol and microsomal type 2 activity with oestradiol and testosterone increased from week 7 to week 20. Activities at 17-20 weeks approximated those at 38-40 weeks. The high, cytosolic oestradiol/T activity ratio (160 +/- 20), characteristic of 17-HOR type 1, was constant between weeks 7 and 20, as was the low microsomal ratio (3.4 +/- 0.4) characteristic of the type 2 activity. There was a relationship between cytosolic type 1 activity and microsomal type 2 activity between weeks 7 and 20 (r = 0.59, P = 0.0055). These results indicate both activities increase coincident with the luteal-placental shift and that their temporal patterns of development are related between weeks 7 and 20 of gestation.

Gene expression of 17 beta-hydroxysteroid dehydrogenase type 2 isozyme in primary cultures of human trophoblasts predicts different mechanisms regulating type 1 and type 2 enzymes.

Two 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) genes, types 1 and 2, have been cloned. The two isozymes show a 30% sequence homology but differ markedly in their kinetic properties. To date, the steroidogenic capacity of the placenta has been associated with syncytium formation. In this study, we have investigated 17 beta-HSD type 1 and type 2 gene expression during trophoblast differentiation in culture. We observed that term placenta and fetal cotyledons contain large amounts of both messenger RNAs (mRNAs). In culture, the type 1 gene is expressed concurrent with syncytium formation. However, type 2 expression is barely detectable in freshly isolated cytotrophoblasts and undetectable in syncytiotrophoblasts. Incubation of trophoblasts with progesterone and estradiol increased type 1 mRNA but did not restore 17 beta-HSD type 2 expression. 17 beta-HSD activities with substrates that differentiate the type 1 and type 2 enzymes correlated with the gene expression results. Type 1 activity decreased in freshly isolated trophoblasts by 2-fold and remained at these levels throughout the culture period. However, when compared with levels measured in term microsomes, type 2 activity decreased by 20-fold in freshly isolated cells and decreased again in culture by 5-fold. The expression pattern of 17 beta-HSD type 1 and type 2 activity in trophoblasts in culture suggests differing mechanisms regulate type 1 and type 2 mRNA levels.

A comparison of 17 beta-hydroxysteroid oxidoreductase type 1 and type 2 activity of cytosol and microsomes from human term placenta, ovarian stroma and granulosa-luteal cells.

A large body of evidence suggests multiple forms of 17 beta-hydroxysteroid oxidoreductase (17-HOR) regulate estrogen and androgen levels within gonadal and peripheral tissues. Two kinetically-differing 17-HOR activities have been detected in placental homogenates. 17-HOR type 1, found mainly in the cytosol, is highly reactive with estradiol-17 beta (E2) and estrone (E1) but not testosterone (T) (high E2/T activity ratio). Microsomal 17-HOR type 2 is reactive with both E2 and T (low E2/T activity ratio). In this study, 17-HOR activity of cytosol and microsomes from term placenta, ovarian stroma and granulosa-luteal cells was assayed under conditions which specifically differentiate between the two forms of the enzyme. Placenta had the highest activity with either E2 or T in both cytosol and microsomes and stroma the lowest. The highest specific activity with E2 and E1 was cytosolic in all samples. The highest specific activity with T was microsomal in placenta and ovarian stroma. E2/E1 activity ratios were comparable for cytosol and microsomes while E2/T activity ratios were comparable for placenta and stroma, but markedly elevated in granulosa-luteal (G-L) cell cytosol and microsomes. The results indicate trophoblast and ovarian stroma have more 17-HOR type 2 relative to type 1. G-L cells, in contrast, are relatively enriched in 17-HOR type 1 and thus have a greater capacity for net conversion of E1 to E2 under physiologic conditions. These differences may contribute to increasing serum and follicular fluid E2/E1 ratios during development of the dominant follicle.

Placental 17 beta-hydroxysteroid oxidoreductase, lactate dehydrogenase and malate dehydrogenase during the latter half of pregnancy in the mouse.

The specific activity of 17 beta-hydroxysteroid oxidoreductase (17-HOR) with estradiol-17 beta (E2), estrone (E1) and testosterone (T), as well as that of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) were measured in homogenates of CF-1 mouse placenta during the latter half of pregnancy. 17-HOR activity with E2 and T increased over 100-fold between days 9 and 12, and 3- to 4-fold between days 15 and 19, with no further change to day 21. In contrast, activity with E1 increased 39-fold between days 9 and 12, 3.8-fold between days 15 and 19 but then decreased between days 19 and 21. The E2/T activity ratio was constant while the E2/E1 ratio increased between days 9 and 21. LDH increased 2-fold between days 9 and 12 with no further increase to day 19. MDH was constant from day 9 to 19. Activity with E2 was inhibited by T, 5 alpha-dihydrotestosterone (5 alpha-DHT) and DHA but not by E1, androstenedione (A) or 20 alpha-dihydroprogesterone. Activity with T was inhibited by E2, 5 alpha-DHT and DHA, but not by A. In contrast, activity with E1 was inhibited by A and DHA but not by E2, T or 5 alpha-DHT. The results suggest placental 17-HOR is developmentally regulated. Although the results are also suggestive of multiple forms of 17-HOR, a single enzyme with an ordered kinetic mechanism cannot be ruled out.

3 beta-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5 alpha-androstane-3 beta,17 beta-diol and dehydroepiandrosterone as substrates.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of human placental 17 beta-hydroxysteroid oxidoreductase: mechanism of stimulation of 17 beta-estradiol formation from estrone by 5 alpha-dihydrotestosterone in homogenates and villi in vitro.

The mechanism of stimulation of 17 beta-estradiol (E2) formation from estrone (E1) by 5 alpha-dihydrotestosterone (5 alpha-DHT) in placental villi was investigated by examining; (1) if dehydroepiandrosterone (DHA) was stimulatory, (2) if NAD(P)H-generating, non-steroidal substrates stimulated E2 formation, (3) the subcellular localization of the effect, (4) if NAD(P) or NAD(P)H was required and (5) rates of 5 alpha-DHT oxidation by villi and microsomes. Although 5 alpha-DHT and DHA both inhibited the E2 to E1 reaction in villi and microsomes, only 5 alpha-DHT stimulated the conversion of E1 to E2. Glucose and lactate were slightly stimulatory when compared with 5 alpha-DHT. Stimulation of E2 formation was observed with microsomes but not with cytosol, and NAD or NADP was required. The results indicate that neither inhibition of the back reaction, E2 to E1, nor NADH or NADPH formation via the 3 beta-hydroxysteroid dehydrogenase/5-ene-3-ketosteroid isomerase reaction can account for the stimulation. It is proposed that the mechanism of stimulation involves one or more forms of membrane-bound 17 beta-hydroxysteroid oxidoreductase with NADH or NADPH formed as a product of 5 alpha-DHT oxidation being used as the cofactor for E1 reduction. This may involve a direct transfer of reduced pyridine nucleotide between enzyme molecules without equilibration with intracellular coenzyme pools.

Pyridine nucleotide levels under conditions of 5 alpha-dihydrotestosterone-stimulated 17 beta-estradiol formation from estrone and pathway of nicotinamide adenine dinucleotide biosynthesis in placental villi in vitro.

It has been reported that testosterone, 5 alpha-dihydrotestosterone and 20 alpha-dihydroprogesterone, substrates for pyridine nucleotide-dependent hydroxysteroid oxidoreductases, stimulate the conversion of estrone to 17 beta-estradiol (E2) by placental villi in vitro. Their enzyme-catalyzed oxidation generates either NADH or NADPH. If the concentration of either reduced nucleotide were rate determining in the conversion of estrone to E2, then increases in NADH or NADPH levels as the result of steroid oxidation could stimulate E2 formation. In this investigation, enzymatic assays were used to quantitate NAD, NADP, NADH, and NADPH in villous tissue from term placenta under conditions where E2 formation was stimulated by 5 alpha-dihydroxytestosterone. On the basis of concurrent observations that NAD levels varied initially and decreased in tissue samples in culture over a 24-h period, the ability of villi to incorporate [14C]nicotinic acid or [14C]nicotinamide into NAD was also examined. No changes were detected in the ratios of oxidized to reduced [14C]nicotinamide nucleotides under stimulatory conditions. NAD was formed only from nicotinic acid. The data suggest that NAD and NADP reduction, if it is the basis for stimulation, is tightly coupled to reoxidation. It would also appear that media used widely for villous tissue and cell culture may not be optimal for pyridine nucleotide synthesis.

Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides.

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.

Steroid modulation of 17 beta-hydroxysteroid oxidoreductase activities in human placental villi in vitro.

Recent data are consistent with the presence of two 17 beta-hydroxysteroid oxidoreductase (17 beta-HOR) activities in placental homogenates. One is localized to intracellular membrane fractions. The conversion of 17 beta-estradiol (E2) to estrone (E1) by this enzyme can be completely inhibited in vitro by C19 and C21 steroids. The second activity is detected in microsomes, but is recovered principally in the cytosol. It also has 20 alpha-HOR activity, but has a high affinity only for C18 steroids. We used this difference to estimate the relative contributions of the two enzymes to E2, E1, and testosterone (T) metabolism by villous tissue in vitro. Fragments of tissue from vaginally delivered placentas (38-40 weeks) were incubated with [3H]E2, [3H]E1, or [3H]T as substrates and various unlabeled steroids as potential inhibitors. Approximately 40% of the E2 to E1 reaction was not inhibited by C19 steroids at 100-200 microM, whereas the conversion of T to androstenedione was inhibited by 90% or more under similar conditions. In contrast, the metabolizable C19 and C21 steroids, 5 alpha-dihydrotestosterone and 20 alpha-dihydroprogesterone, which inhibited the conversion of E1 to E2 by microsomes, stimulated E2 formation from E1 by villi. We conclude that nonspecific 17 beta-HOR accounts for approximately 60% of the E2 to E1 conversion and nearly all of the T to androstenedione conversion in villous tissue fragments. The data also suggest that net E2 formation in villi is catalyzed principally by the C18-specific 17 beta-HOR.

Mannitol pharmacokinetics and serum osmolality in dogs and humans.

The relationship between mannitol pharmacokinetics and changes in serum osmolality were studied in dogs and humans. Four human subjects each received between 0.5 and 0.7 g/kg of mannitol as an i.v. infusion given over 15 min. Intravenous bolus doses of 0.5, 1.0 and 1.5 g/kg were given to each of five animals. Serial determinations of serum osmolality and serum mannitol concentrations were then performed. Mannitol disposition was best described using a biexponential equation and assuming a two-compartment, open model with elimination from the central compartment. For human subjects, the mean (+/- S.D.) distribution half-life was 2.11 +/- 2.67 min and the elimination half-life was 71.15 +/- 27.02 min. The volume of distribution was 0.47 +/- 0.50 liters/kg and total body clearance was 7.15 +/- 10.23 ml X min-1 X kg-1. The disposition of mannitol in dogs is similar to that observed in humans. Mannitol clearance was independent of dose whereas the central compartment volume was significantly larger (P less than .005) in animals receiving the 1.5-g/kg dose. The volume change is probably due to a rapid, uncompensated shift of water from intracellular to extracellular space. There was a strong positive correlation (r = 0.90) between mannitol concentration and serum osmolality changes. However, neither maximum serum mannitol concentration nor maximum serum osmolality increased proportionately with dose. Only the 1.5-g/kg dose produced a sustained elevation of serum osmolality, confirming that larger doses are more likely to result in prolonged hypertonic dehydration.

Fetal lung maturity assessment by a modified A650 determination.

This paper presents an evaluation of a modified absorbance method for estimating fetal lung maturity. Absorbance at 650 nm in combination with a two-step centrifugation procedure was used in an attempt to focus more directly on lamellar bodies and evaluate the contribution of residual absorbance due to non-lamellar body materials. Absorbance values after centrifugation at 250 X g for 5 minutes (A250) and 10,000 X g for 20 minutes (A10,000) were taken as estimates of total absorbance due to lamellar bodies plus non-lamellar body material and that due to non-sedimentable, non-lamellar body material respectively. These values were used to generate two new parameters: delta A (A250-A10,000), to better estimate absorbance due to lamellar bodies, and %A (delta A/A250 X 100), to express lamellar body absorbance in terms of total observable absorbance and thereby minimize effects of dilution. The three parameters (A250, delta A, %A) were used in combination to create a battery (ABatt) of absorbance values for each fluid sample. Absorbance after centrifugation at 2,000 X g for 10 minutes (A2,000), a widely used standard method, was also evaluated for purposes of comparison. A250 was designated as mature if greater than or equal to 0.350, delta A was called mature if greater than or equal to 0.250, and %A was considered mature if greater than or equal to 75%. If any of the parameters was immature, ABatt was called immature. The range of values for A250, A2,000, and A10,000, increased with gestational age in the total population as well as the corrected population (excluding amniotic fluid contaminants, and pregnancies with isoimmunization or diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)