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Biomedical subjects

L Milewich

Publications and source records attributed to L Milewich.

At least 55 records · Page 3Linked to original sources
PubMed1985

18-Substituted steroids: synthesis of 18-hydroxycortisol (11 beta,17 alpha,18,21-tetrahydroxy-4-pregnene-3,20-dione) and 18-hydroxycortisone (17 alpha,18,21-trihydroxy-4-pregnene-3,11,20-trione).

The isolation of 18-hydroxycortisol from the urine of patients with primary aldosteronism was recently described and no synthetic procedure was available for its preparation. The C-13 angular methyl group of prednisolone-17 alpha,21-acetonide-11 beta-nitrite was functionalized by photolysis in the presence of oxygen to give the product 18-hydroxy-prednisolone-17 alpha,21-acetonide-18-nitrate. The 18-nitrate was reduced with zinc and ammonium acetate to the corresponding 18-hydroxy compound, 18-hydroxy-prednisolone-17 alpha,21-acetonide. Homogeneous hydrogenation with Tris(triphenyl-phosphine)rhodium (I) chloride as catalyst resulted in the formation of 18-hydroxy-cortisol-17 alpha,21-acetonide. Acid hydrolysis of the latter compound gave 18-hydroxycortisol. Oxidation of 18-hydroxycortisol-17 alpha,21-acetonide with pyridinium dichromate followed by acid hydrolysis gave 18-hydroxycortisone. The 18-hydroxylated steroids exist as the 18,21-hemiacetals. Catalytic reduction with tritium gas with Tris(triphenyl-phosphine)rhodium (I) chloride of 18-hydroxyprednisolone-17 alpha,21-acetonide and acid hydrolysis gave [1,2(3)H]18-hydroxycortisol.

Chemical Phenomena↗
PubMed1985

17 beta-Hydroxysteroid oxidoreductase: a ubiquitous enzyme. Interconversion of estrone and estradiol-17 beta in BALB/c mouse tissues.

A survey was conducted to define the sites of 17 beta-hydroxysteroid oxidoreductase activity in organs and tissues of male and female BALB/c mice, as well as the favored direction of the oxidoreductase reaction in intact tissues. The enzyme activity was assayed by use of radiolabeled estrone and estradiol-17 beta as substrates. Estrone formation from estradiol-17 beta was demonstrated in all tissues. The formation of estradiol-17 beta from estrone was demonstrated in most tissues, however, it was barely detected or was undetectable in the glandular stomach, small intestine, cecum, and large intestine. Thus, 17 beta-hydroxysteroid oxidoreductase activity is expressed in all BALB/c mouse organs and tissues. Approximately two-thirds of the tissues and organs examined, including those of the reproductive tracts, favored the conversion of estrone to estradiol-17 beta rather than the reverse reaction. The results of this study, however, represent qualitative estimates of 17 beta-hydroxysteroid oxidoreductase activity in BALB/c mouse tissues that are uncorrected for conversion to hydroxylated metabolites. These in vitro findings suggest that the 17 beta-hydroxysteroid oxidoreductase catalyzed reduction of estrone may contribute to the maintenance of physiologic levels of estradiol-17 beta in estrogen responsive tissues.

17-Hydroxysteroid Dehydrogenases↗
PubMed1985

Origin of urinary nonconjugated 19-nor-deoxycorticosterone and metabolism of infused radiolabeled 19-nor-deoxycorticosterone in men and women.

It is known that 19-nor-deoxycorticosterone (19-nor-DOC) is a potent mineralocorticosteroid that is present in urine of rats and humans in a free, i.e., nonconjugated, form. In some forms of hypertension in rats, the levels of free 19-nor-DOC in urine are increased compared with those in urine of normotensive animals. Yet, despite the potential importance of this mineralocorticosteroid in the pathogenesis of certain forms of hypertension, little is known of its site of origin or metabolism. In the present investigation, we evaluated the metabolism of intravenously infused [3H]19-nor-DOC and the possibility that 19-nor-DOC was formed from plasma DOC. We found that the metabolism of [3H]19-nor-DOC infused intravenously in men and women was similar to that of DOC with important exceptions. The majority of the radiolabeled urinary metabolites of intravenously infused [3H]19-nor-DOC were excreted in urine as glucuronosides. Little radioactivity, infused as [3H]19-nor-DOC, was recovered in urine as nonconjugated or sulfoconjugated steroids. There was no free radiolabeled 19-nor-DOC in urine after the simultaneous infusion of [3H]19-nor-DOC and [14C]DOC. A major metabolite of [3H]19-nor-DOC in urine was 19-nor-DOC-21-glucuronoside, whereas little or no intravenously infused radiolabeled DOC was excreted as radiolabeled DOC-glucuronoside. We also found that intravenously infused [14C]DOC was not converted to urinary [14C]19-nor-DOC (glucuronoside) and that other tritium-labeled metabolites of infused [3H]19-nor-DOC contained no carbon-14. The production rate of 19-nor-DOC, computed from the specific activity of urinary 19-nor-DOC (glucuronoside), in one normal man was 16 micrograms/d and in the two women of this study, it was 10 micrograms/d. These findings are supportive of the proposition that free urinary 19-nor-DOC is not formed from plasma DOC; it may be formed in kidney from a precursor other than DOC or it may be formed nonenzymatically in kidney or urine from a precursor such as 19-oic-DOC.

Carbon Radioisotopes↗
PubMed1985

Metabolism of androsterone and 5 alpha-androstane-3 alpha,17 beta-diol in human lung tissue and in pulmonary endothelial cells in culture.

The metabolism of [3H]androsterone and [3H] 5 alpha-androstane-3 alpha,17 beta-diol ( [3H] 3 alpha-diol) was studied in slices of human lung tissue and cultures of human pulmonary artery endothelial cells. Lung tissue metabolized [3H]androsterone (0.25 microM) to 5 alpha-androstane-3,17-dione (30.3 pmol 100 mg-1 tissue h-1), isoandrosterone (0.7 pmol 100 mg-1 tissue h-1), 5 alpha-dihydrotestosterone (5 alpha-DHT; 0.1 pmol 100 mg-1 tissue h-1), 3 alpha-diol (0.1 pmol 100 mg-1 tissue h-1), and two polar metabolites. Pulmonary arterial endothelial cells produced the same metabolites of [3H]androsterone (0.083 microM), with the exception of the polar compounds [5 alpha-androstane-3,17-dione (1.3 pmol mg-1 protein h-1), isoandrosterone (0.1 pmol mg-1 protein h-1), 5 alpha-DHT (0.2 pmol mg-1 protein h-1), and 3 alpha-diol (0.2 pmol mg-1 protein h-1)]. Thus, the principal metabolite of [3H]androsterone in both lung tissue and endothelial cells was 5 alpha-androstane-3,17-dione. Human lung tissue metabolized [3H]3 alpha-diol (0.28 microM) to 5 alpha-DHT (8.8 pmol 100 mg-1 tissue h-1), androsterone (2.2 pmol 100 mg-1 tissue h-1), 5 alpha-androstane-3,17-dione (0.8 pmol 100 mg-1 tissue h-1), isoandrosterone (0.1 pmol 100 mg-1 tissue h-1), and four polar metabolites (0.2 pmol 100 mg-1 tissue h-1). 5 alpha-DHT was the principal metabolite of [3H]3 alpha-diol within the first hour of incubation, but the concentration of this androgen declined thereafter to 3.6 pmol 100 mg-1 tissue after 4 h of incubation. This decline was correlated with increased 5 alpha-androstane-3,17-dione synthesis (6.7 pmol 100 mg-1 tissue 4 h-1). Androsterone formation from [3H]3 alpha-diol, however, was linear with time of incubation for 4 h (8.9 pmol 100 mg-1 tissue 4 h-1). The formation of these products demonstrates that the principal 5 alpha-reduced-C19-steroid-metabolizing enzymes in human lung are 3 alpha-hydroxysteroid oxidoreductase.

Adolescent↗
PubMed1985

Steroid sulfatase activity in amnion tissue and amnion cells maintained in monolayer culture.

Phenolic steroid sulfatase activity in amnion tissue, amnion homogenate, subcellular fractions, and amnion epithelial cells in culture was demonstrated with radiolabeled estrone sulfate as the substrate. Sulfatase activity could not be detected in either amnion tissue or cells when evaluated with dehydroisoandrosterone sulfate as the substrate. Phenolic steroid sulfatase activity in amnion tissue was linear with incubation time up to 3 h and with amnion tissue weight up to 800 mg/ml. The rate of estrone sulfate hydrolysis in amnion tissue increased in a linear manner with temperature from 3 to 60 C. The apparent Km of amnion tissue sulfatase for estrone sulfate was 9 microM. The highest specific activity of the enzyme was found in both the mitochondrial-lysosomal and microsomal fractions. In studies with amnion epithelial cells in monolayer culture, phenolic steroid sulfatase activity was linear with incubation time up to 4 h and with cell number up to 2 X 10(5)/ml. The apparent Km of amnion cell sulfatase for estrone sulfate was 5.5 microM. The product of hydrolysis, i.e. estrone, was metabolized in situ to 17 beta-estradiol in both amnion tissue and cells. The hydrolysis of estrone sulfate (and possibly other phenolic steroid sulfates present in amniotic fluid) by amnion cells may be important in providing biologically potent estrogens for in situ action.

Amnion↗
PubMed1984

Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues.

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Conversion of androstenedione to testosterone and other androgens in guinea-pig alveolar macrophages.

Alveolar macrophages obtained by bronchoalveolar lavage of lungs of male and female guinea pigs were incubated with tritium-labelled androstenedione to evaluate the steroid metabolizing enzymes in these cells. The radiolabeled metabolites were isolated and thereafter characterized as testosterone, 5 alpha-androstanedione, 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol. Thus, the following androstenedione metabolizing enzymes are present in guinea-pig alveolar macrophages: 17 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase, 3 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase. The predominant androstenedione metabolizing enzyme activity present in alveolar macrophages was 17 beta-hydroxysteroid dehydrogenase. The rate of testosterone formation increased with incubation time up to 4 h, and with macrophage number up to 1.6 X 10(7) cells per ml. Androstenedione metabolism was similar in alveolar macrophages obtained both from male and female guinea pigs. These results suggest that alveolar macrophages may be a site of peripheral transformation of blood-borne androstenedione to biologically potent androgens in vivo and, therefore, these cells may contribute to the plasma levels of testosterone in the guinea pig.

Androgens↗
PubMed1983

Androstenedione metabolism in human alveolar macrophages.

The metabolism of [3H]androstenedione by human alveolar macrophages was investigated. Alveolar macrophages were obtained by bronchopulmonary lavage by use of a heparinized saline solution devoid of Ca++ and Mg++. After purification, the macrophages were incubated at 37 C in RPMI-1640 medium that contained glucose and [1,2,6,7-3H]androstenedione under various experimental conditions. Control incubations were conducted without macrophages. After incubation, 14C-labeled steroids that corresponded to the metabolites were added as internal recovery standards. The metabolites were characterized by chromatography and crystallization to constant 3H to 14C ratios. Human alveolar macrophages convert [3H]androstenedione to 5 alpha-androstane-3,17-dione, testosterone, 5 alpha-dihydrotestosterone, androsterone, and isoandrosterone. Unidentified polar metabolites also were formed. Therefore, the following enzymes are present in these cells: 5 alpha-reductase, 17 beta-hydroxysteroid dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, and unknown hydroxylase(s). The rates of formation of the principal metabolites, 5 alpha-androstanedione and testosterone, remained linear up to 4 h of incubation and with macrophage number up to 1.5 X 10(7) cells/ml. These findings suggest that alveolar macrophages may be involved in the peripheral metabolism of androstenedione to potent androgens in man. It is possible that androgens, formed from blood-borne androstenedione within alveolar macrophages, may modulate phagocytic and other activities in these cells.

Androstenedione↗
PubMed1983

Metabolism of dehydroisoandrosterone and androstenedione in human pulmonary endothelial cells in culture.

The capacity of endothelial cells from pulmonary arteries and veins to convert dehydroisoandrosterone (3 beta-hydroxy-5-androsten-17-one) and androstenedione to potent, biologically active steroids was investigated. The metabolites of [3H]dehydroisoandrosterone produced in pulmonary artery endothelial cells were androstenedione and 5-androstene-3 beta, 17 beta-diol. The metabolites isolated from incubation of pulmonary arterial cells with [3H]androstenedione were testosterone, 5 alpha-androstane-3,17-dione, 5 alpha-dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), isoandrosterone (3 beta-hydroxy-5 alpha-androstan-17-one), and androsterone. The products of [3H]androstenedione metabolism in human pulmonary venous cells were the same as those formed in arterial cells, and in addition, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol were formed. The rates of metabolite formation from [3H]androstenedione in pulmonary arterial and venous endothelial cells were linear with incubation time up to 3 h. These findings suggest that the pulmonary endothelium is an important site for the metabolism of dehydroisoandrosterone and androstenedione in the human lung. Endothelial cells produce the same metabolites as human lung tissue, with the exception of hydroxylated steroids.

Androstenediol↗
PubMed1983

Steroid sulfatase activity in human lung tissue and in endothelial pulmonary cells in culture.

The conversion of tritium-labeled estrone sulfate to [3H]estrone was evaluated in human lung tissue in vitro. Under standardized conditions, the rate of hydrolysis of [3H] estrone sulfate to [3H]estrone was linear with time of incubation up to 4 h and with wet tissue weight up to 400 mg/ml. The apparent Km of sulfatase for estrone sulfate was 9 microM, and the maximum velocity was 1.4 nmol substrate hydrolyzed/100 mg lung . h. The lung tissue also metabolized the primary metabolite of [3H]estrone sulfate, [3H]estrone, to 17 beta-[3H]estradiol. The hydrolysis of [3H]dehydroisoandrosterone sulfate to [3H]dehydroisoandrosterone by human lung tissue was also measured. Sulfatase activity with this substrate was linear as a function of wet tissue weight up to 800 mg/ml. The apparent Km of sulfatase for dehydroisoandrosterone sulfate was 7 microM, and the maximum velocity was 1.0 nmol substrate hydrolyzed/100 mg lung . h. The highest specific activity of lung sulfatase for [3H]dehydroisoandrosterone sulfate was found in a microsomal fraction of lung homogenate. The primary metabolite, [3H]dehydroisoandrosterone, was metabolized further by lung tissue to [3H]androstenedione and [3H]5-androstene-3 beta, 17 beta-diol. Although isolated segments of human pulmonary arteries also metabolized both [3H] estrone sulfate and [3H]dehydroisoandrosterone sulfate, cultures of pulmonary arterial endothelial cells lacked sulfatase activity. The cell(s) source of sulfatase activity in human lung tissue and isolated arteries has not yet been identified. Our findings suggest that the metabolism of sulfated steroids by the lung should be considered in evaluating homeostasis.

Cells, Cultured↗
PubMed1982

Androstenedione metabolism by human lymphocytes.

Intact human lymphocytes were incubated with tritium-labeled androstenedione; after extraction and appropriate chromatographic separation, followed by crystallization to constant tritium to carbon-14 ratio to establish radiochemical homogeneity, the following metabolites were identified: testosterone, 5 alpha-androstane-3,17-dione, 5 alpha-dihydrotestosterone, androsterone and isoandrosterone. Neither estrogen nor 5 beta-reduced metabolites were found as products. The major androstenedione metabolizing activities in human lymphocytes were 5 alpha-reductase and 17 beta-hydroxysteroid oxidoreductase; in addition, 3 alpha-and 3 beta-hydroxysteroid oxidoreductase activities also were present in these cells. The rates of metabolite formation were linear with incubation time up to 4 h, and with cell number up to 2 x 10(7) lymphocytes per ml for the 5 alpha-reduced products and up to 1.6 x 10(7) lymphocytes per ml for testosterone. These findings are suggestive that, in the human, lymphocytes are a potential site of peripheral metabolism of androstenedione to potent adnrogens.

Androstenedione↗
PubMed1982

Interconversion of estrone and estradiol-17 beta in lung slices of the adult human.

The metabolism of tritium-labeled estrone and estradiol-17 beta in slices of lung tissue obtained from an adult human was studied; estrone was identified as the only metabolite of estradiol-17 beta and estradiol-17 beta as the exclusive product of estrone metabolism. Product formation remained linear as a function of time of incubation up to 3 h and of wet lung tissue mass up to 300 mg/ml. At equimolar substrate concentrations, the rates of estrone formation were at least 2-fold greater than those of estradiol-17 beta. The apparent KM of 17 beta-hydroxysteroid oxidoreductase for estrone was 11 microM and that for estradiol-17 beta was 10 microM. These results are suggestive that the human lung enzyme binds estrone and estradiol-17 beta with similar affinities; however, the oxidative pathway is favored as indicated by the greater Vmax attained in the formation of estrone. It is possible that, in vivo, the human lung constitutes a site for estradiol-17 beta inactivation to estrone as well as a site for the conversion of estrone to estradiol-17 beta. This last process may become particularly important in instances in which the ovaries have ceased to function and secrete estradiol-17 beta, e.g. the postmenopausal women.

Carcinoma, Squamous Cell↗
PubMed1982

Metabolism of androstenedione by guinea-pig peritoneal macrophages: synthesis of testosterone and 5 alpha-reduced metabolites.

Androstenedione metabolizing enzymes present in guinea-pig peritoneal macrophages were investigated using tritium-labeled androstenedione as the substrate. We found that the metabolites of [3H]-androstenedione produced by these macrophages were testosterone, 5 alpha-androstane-3,17-dione, isoandrosterone, androsterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol. The rates of metabolite formation remained linear as a function of time of incubation for approximately 30 min and with macrophage number up to 2 x 10(7) cells per ml. The formation of these metabolites in indicative that the following androstenedione metabolizing enzymes are present in guinea-pig peritoneal macrophages: 5 alpha-reductase, 3 alpha-hydroxysteroid oxidoreductase, 3 beta-hydroxysteroid oxidoreductase and 17 beta-hydroxysteroid oxidoreductase. It is possible, therefore, that the macrophage, in vivo, may play a role in the metabolism of blood-borne androstenedione to potent androgens. These hormones are important in the regulation of many biological processes, possibly including the activity of the macrophage itself.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1982

Identification of 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone as products of deoxycorticosterone metabolism by rat adrenals.

The formation of 19-hydroxydeoxycorticosterone (19,21-dihydroxy-4-pregnen-3,20-dione), 19-oxo-deoxycorticosterone (21-hydroxy-4-pregnen-3,19,20-trione), and 19-oic-deoxycorticosterone (19-oic-21-hydroxy-4-pregnen-3,20-dione) from precursor deoxycorticosterone by adrenal glands obtained from intact rats and from rats undergoing adrenal regeneration was demonstrated. These metabolites were isopolar with corresponding authentic steroid standards on thin layer chromatography, gas chromatography, and high pressure liquid chromatography. They were further characterized by either mass spectrometry or gas chromatography-mass spectrometry. Therefore, rat adrenals have the enzymes required to convert deoxycorticosterone to 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone; however, rat adrenals do not convert deoxycorticosterone or any of the oxygenated metabolites to 19-nor-deoxycorticosterone (21-hydroxy-19-nor-4-pregnen-3,20-dione). It is possible, however, that 19-nor-deoxycorticosterone is formed at peripheral sites from the oxygenated deoxycorticosterone precursors.

Adrenal Glands↗
PubMed1982

Metabolism of androstenedione by human platelets: a source of potent androgens.

The metabolism of tritium-labeled androstenedione by human platelets was studied in vitro. The following metabolites were identified: testosterone, 5 alpha-androstane-3,17-dione, androsterone, isoandrosterone, 5 alpha-dihydrotestosterone, and 5 alpha-androstane-3 alpha,17 beta-diol. The rates of formation of these metabolites remained linear with time of incubation up to 2 h and with increased platelet protein concentration up to 14.6 mg/ml. Estrogens and 5 beta-reduced metabolites were not formed by platelets. The major enzyme systems involved in the metabolism of androstenedione in human platelets were the 17 beta-hydroxysteroid oxidoreductase and 5 alpha-reductase activities; in addition, 3 alpha-and 3 beta-hydroxysteroid oxidoreductase activities were also present in these megakaryocyte fragments. Thus, it appears that platelets are a potential site of extraglandular conversion of androstenedione to potent androgens in man. It is also possible that androgens formed within the platelet may affect platelet function by regulating their sensitivity to aggregating stimuli.

Adult↗