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Biomedical subjects

J M Larner

Publications and source records attributed to J M Larner.

At least 37 records · Page 2Linked to original sources
PubMed1992

Glomus jugulare tumors. Long-term control by radiation therapy.

The records of 49 patients with glomus jugulare tumor seen at the University of Virginia from 1932 to 1985 were retrospectively reviewed with the objective of assessing long-term results of treatment. Follow-up ranged from 5 to 31 years, with a minimum of 10 years in 36 patients (73%). According to McCabe's classification, 17 patients (35%) were Group I, 11 patients (22%) were Group II, and 21 patients (43%) were Group III. Analysis by therapeutic technique revealed that 20 patients received surgery alone (41%), 15 patients received radiation therapy alone (31%), and 14 patients received combined therapy (28%). Only 7 patients (14%) have had clinical or radiologic evidence of disease progression. Three of these patients were treated by surgery alone and three by a combination of surgery and radiation, but the dose was less than 4000 cGy. Only one patient treated by radiation therapy alone or with surgery and radiation to a dose in excess of 4000 cGy demonstrated disease progression. Salvage radiation therapy was given to three of the seven patients when progressive disease was detected. Radiation therapy is an effective treatment for glomus jugulare tumor with minimal late progression of disease in adequately treated patients and no significant long-term complications.

Adolescent↗
PubMed1992

Aromatase and testosterone fatty acid esters: the search for a cryptic biosynthetic pathway to estradiol esters.

The estradiol fatty acid esters (lipoidal derivatives, LE2) are extremely potent estrogens that accumulate in fat, including fat of menopausal women. These steroidal esters are protected from metabolism and are converted to the free, biologically active steroid through the action of esterases. Previous studies have shown that biosynthetic pathways in the adrenal gland exist in which steroid fatty acid esters are substrates. This led us to determine whether a cryptic aromatase pathway exists in which testosterone esters could be converted directly into LE2. We tested a representative fatty acid ester, testosterone stearate, both as an inhibitor and as a substrate for the aromatase enzyme from human placental microsomes. This ester had neither activity. In addition, we tested [1 beta-3H]testosterone acetate as a substrate for this enzyme complex, measuring the production of 3H2O as evidence of aromatization. Although the rate of reaction was considerably slower than that of testosterone, 3H2O was produced. However, when [2, 4, 6, 7-3H]testosterone acetate was incubated and the steroidal products isolated, we found that hydrolysis of the substrate had occurred. Both [3H]-labeled testosterone and estradiol were found, and very little if any [3H]estradiol acetate was formed. Thus, we conclude that an aromatase pathway involving testosterone esters does not exist and that the sole source of LE2 is through direct esterification of estradiol.

Aromatase↗
PubMed1992

Measurement of estradiol-17-fatty acid esters in human tissues.

We have developed a gas chromatographic/mass spectral method for the sensitive and reproducible measurement of estradiol-17-fatty acid esters in human tissues and blood. To provide an internal standard for quantification, a trideuterated analog of a representative estradiol ester is added to the tissues. Estradiol (E2) released from the nonpolar ester fraction by alkaline hydrolysis is derivatized to form the ditrimethylsilyl ether and then analyzed by gas chromatographic/mass spectral, monitoring the molecular ions mass per U charge of the ditrimethylsilyl derivative of E2 and [2H3]E2. There are low but detectable levels of E2 ester in the blood of cycling females; there are none in urine. While the E2 ester is present in breast cyst fluid, its concentration, 77-140 pmol/L, is considerably less than E2, 110-2,863 pmol/L. But there is a large amount of E2 ester in fat. In premenopausal women the average E2 ester in fat (sc and omental) is 957 +/- 283 38 fmol/g (SEM); in women who are menopausal less than 12 yr, the E2 ester in fat is 669 +/- 158 fmol/g; in women who are menopausal at least 15 yr, the fat level is 399 +/- 146 fmol/g. Muscle from the same women have lower concentrations of the ester; in 8 out of 12 muscle specimens it was not detectable. The E2 esters are extremely potent estrogens. Although they are hormonally active they require enzymatic hydrolysis to exert their hormonal action. These studies show that these long chain esters of E2 are sequestered in fatty tissues, wherein they represent a protected store of preformed hormone. Under the proper stimulation, adipose tissue can activate the estrogenic signal through the action of hormonally sensitive esterases. Thus, through signaling between estrogen sensitive tissues and neighboring fat cells, a local paracrine loop may exist.

Adipose Tissue↗
PubMed1991

Steroidal fatty acid esters.

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3 beta-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3 beta-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estradiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 alpha and C-17 beta. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.

Adrenal Glands↗
PubMed1991

Estrogenic action of estriol fatty acid esters.

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.

Alkaline Phosphatase↗
PubMed1991

Phase I/II trial of superselective arterial 5-FU infusion with concomitant external beam radiation for patients with either anaplastic astrocytoma or glioblastoma multiforme.

From August 1987 through September 1989, 25 patients with either anaplastic astrocytoma (8 patients) or glioblastoma multiforme (17 patients) were entered into a Phase I trial of combined intra-arterial 5-fluorouracil (5-FU) and external beam radiation therapy. The intra-arterial 5-FU was given in a superselective, supraopthalmic fashion, in escalating doses from 200 mg to 600 mg on a weekly basis during the radiotherapy. Each patient received from 1-4 courses of 5-FU. Radiotherapy consisted of 5,000 rads in 25 fractions given to partial brain fields including the mass and surrounding edema plus a 3-cm margin as defined by computed tomography scan. There were a total of 4 significant acute complications out of a total of 70 infusions. These included 3 ischemic events of which 2 were transient. In addition, 1 patient experienced a cerebral bleed. No patients developed ocular complications. Electroencephalograms (EEGs) were performed immediately before and during 21 intra-arterial infusions. Two patients developed significant EEG changes during intra-arterial infusion, and both of these patients experienced untoward reactions. The remaining patients showed no EEG changes during their infusions. The median survival for patients with glioblastoma multiforme was 15 months. We believe the toxicity of superselective intra-arterial 5-FU infusion combined with external beam radiotherapy is acceptable. This type of treatment deserves further study.

Adult↗
PubMed1990

Biological activity of the fatty acid ester metabolites of corticoids.

Fatty acid esters of all families of steroid hormones are known to exist naturally. While their physiological roles are not clear, the C-17-fatty acid esters of estradiol are extremely potent and unusually long-lived estrogens. Thus, it appeared that increased potency would be a logical consequence of the esterification of all of the active steroid hormones. To test this hypothesis we measured the effect of an ester of corticosterone, corticosterone-21-stearate, on the induction of tyrosine aminotransferase in adrenalectomized rats. Surprisingly, while the ester is active compared to the unesterified corticoid corticosterone, there was no difference in either the magnitude or the duration of the induction of this enzyme. To determine whether the C-21-steroidal ester could itself induce this gluconeogenic response, we tested corticosterone-21-oleate and corticosterone-21-stearate as competitors for the binding of [3H] dexamethasone to the glucocorticoid receptor in rat liver cytosol. Both were poor ligands, with binding affinities of about 4% and more than 1%, respectively, compared to corticosterone. From these results, it is doubtful that the esters could act directly in vivo without prior cleavage of the fatty acid. We measured the rate of hydrolysis of corticosterone-21-stearate and estradiol-17-stearate by rat liver esterases. Corticosterone-21-stearate is hydrolyzed at a much greater rate (10- to 25-fold) than estradiol-17-stearate. Consequently, the difference in both potency and duration of response between the ester of the corticoid and that of the estrogen can be explained by the very rapid rate of conversion of the former into the unesterified form. Since the esterification of the corticoids appears not to be related to an increased biological half-life, as it is in the estrogens, the question remains as to the physiological role that they might play.

Adrenalectomy↗
PubMed1989

Actions of an estradiol-17-fatty acid ester in estrogen target tissues of the rat: comparison with other C-17 metabolites and a pharmacological C-17 ester.

The C-17 fatty acid esters of estradiol (E2) are a unique family of nonpolar estradiol metabolites. They are potent long-acting estrogens that represent the natural analog of the synthetic esters used for estrogen therapy. We measured the uterotropic response and the formation of uterine nuclear estrogen receptors (ERn) produced by iv administration of a representative ester, E2-17-stearate, in comparison to E2, other natural C-17 conjugates of E2, E2-17-glucuronide, and E2-17-sulfate, and the pharmacological ester E2-17-cyclopentylpropionate. While E2-17-stearate produced a sustained and greater uterotropic response compared to E2, the maximal induction of ERn by the ester was only about one third of that induced by a similar dose of E2. However, the induction of ERn by E2-17-stearate was markedly sustained compared to that by E2. Furthermore, the initiation of the ERn response to E2-17-stearate was delayed. Since E2-17-esters do not bind to the ER, this delay is consistent with the requirement for hydrolysis of the esters before interaction with the ER. Neither of the ionic conjugates of E2 (sulfate and glucuronide) produced an increase in ERn concentrations or a uterotropic response. The synthetic ester cyclopentylpropionate, like E2, produced a rapid ERn response and a significantly shorter uterotropic response than the stearate ester. When the induction of ERn by E2-17-stearate was investigated in other target tissues there were no marked differences in the brain, pituitary, and liver. No blood-brain barrier was apparent for the formation of ERn, despite the fact that this steroidal ester circulates in the blood bound to lipoproteins. These findings suggest that this unusual family of steroidal esters has biological properties that differentiate them from other known estrogens, natural and synthetic, in terms of their ability to produce a slow-onset sustained estrogenic stimulus in a variety of different estrogen target tissues.

Animals↗
PubMed1989

The synthesis of 11 beta-methoxy-[16 alpha-123I] iodoestradiol and its interaction with the estrogen receptor in vivo and in vitro.

In order to produce an estrogen receptor mediated imaging agent, we have synthesized 11 beta-methoxy-16 alpha-iodoestradiol labeled with 123I, and have studied its interaction with the estrogen receptor and its distribution in rats and rabbits. This 123I-labeled steroid, 11 beta-methoxy-16 alpha-[123I]iodoestradiol, binds with high affinity, Ka = 6 x 10(9) M-1, and specificity to the estrogen receptor in uterine cytosol. When tested in vivo, this radiolabeled steroid concentrates by a receptor mediated mechanism, in the estrogen target tissue, the uterus, producing very high target to nontarget tissue ratios. The results of these experiments indicate that 11 beta-methoxy-16 alpha-[123I]iodoestradiol may be a useful imaging agent for clinically monitoring and detecting estrogen receptor containing tumors.

Animals↗
PubMed1987

Binding of estradiol-17-fatty acid esters to plasma proteins.

The C-17 fatty acid esters of estradiol are a unique family of long-acting estrogens that circulate in blood. The unusual duration of the estrogenic action of these esters has been shown previously to correlate with their very slow rate of metabolism. However, in striking contrast to their slow rate of metabolism, the clearance of these esters from blood is relatively rapid, not very different from that of estradiol (E2). Studies on the effect of the size of the carboxylic acid moiety on the rates of both metabolism and clearance have suggested that an active process might be involved in the cellular uptake of these circulating esters, and this, in turn, raised the question of how E2-fatty acid esters are transported in blood. The binding of representative E2-17-fatty acid esters to both human and rat plasma proteins known to bind either E2 or fatty acids was investigated. As expected, both E2 and 5 alpha-dihydrotestosterone bound to human sex hormone-binding globulin, whereas none of the E2 esters bound to this human plasma protein. Similarly, E2 bound to rat alpha-fetoprotein (AFP) and unsaturated fatty acids bound to both human and rat AFP, but none of the E2 esters bound to AFP of either species. These steroid esters bind to lipoproteins. Over 85% of a representative ester, E2-17-stearate, partitioned in the lipoprotein fractions of both human and rat serum, while the synthetic short chain ester, E2-17 beta-acetate, like E2 itself, partitioned predominantly in the nonlipoprotein fraction of blood. These results demonstrate the unusual binding of a family of steroid hormones to plasma lipoproteins and open the possibility that they are transported into target cells through the mediation of lipoprotein receptors.

Animals↗
PubMed1986

16 Alpha-[125I]iodo-11 beta-methoxy-17 beta-estradiol: a radiochemical probe for estrogen-sensitive tissues.

We have synthesized an analog of 16 alpha-iodoestradiol, 11 beta-methoxy-16 alpha-iodo-estra-1,3,5-(10)triene-3,17 beta-diol (16 alpha-iodo-11 beta-methoxyestradiol), as a potential radiopharmaceutical for the in vivo imaging of estrogen-sensitive tissues. This steroid was synthesized labeled with 125I by halogen exchange of the stable intermediate 11 beta-methoxy-16 beta-bromo-17 beta-estradiol with Na125I. The halogen exchange reaction produces the radioiodinated steroid with a 65-80% yield in 3 h. This rapid synthesis and purification of the 125I-labeled estrogen permits a similar synthesis with 123I, a radioisotope with excellent properties for imaging. The 11-methoxy analog is a highly potent estrogen that binds to the estrogen receptor with an affinity equal to that of estradiol. In vivo, 11 beta-methoxy-16 alpha-[125I]iodoestradiol concentrates in an estrogen receptor-dependent manner in the uterus, producing remarkably sustained and much higher uterus to blood ratios than 16 alpha-[125I]iodoestradiol. Thus, this radiosteroid shows great promise, both as a research probe of the estrogen receptor and as a clinical tool for the imaging of estrogen-responsive tumors.

Animals↗
PubMed1985

Synthesis of estradiol fatty acid esters by human breast tumors: fatty acid composition and comparison to estrogen and progesterone receptor content.

The estradiol-17 beta-fatty acid esters are non-polar metabolites of estradiol, formed in many tissues, including human breast tumors. It has been shown in the rat that the synthesis of these esters is greatest in those tissues that respond to estrogen stimulation. Thus the possibility was explored that the biosynthesis of the estradiol esters in human breast tumors occurs mainly in those tumors that are estrogen sensitive; and thus that the synthesis of this family of non-polar metabolites of estradiol could be used as an additional marker for the identification of hormonally dependent tumors. However, the conversion of estradiol to the esters did not correlate with other indicators of estrogen responsiveness, the progesterone or estrogen receptors. Interestingly, the composition of the fatty acids in the estradiol-17-esters synthesized in the human tumors was markedly different from those originally identified in the bovine uterus. In the bovine uterus, the esters were predominantly unsaturated, 85%, while in this study the saturated esters were the major component. Since after systemic administration the saturated estradiol-17-esters have been found to be much longer-lived than the unsaturated esters, the biosynthesis of the relatively high proportion of saturated esters by human breast tumors may indicate a significantly prolonged duration for the estrogenic signal produced by endogenously formed estradiol esters. These esters formed and sequestered within the tumor cell, may serve as a preformed store of estradiol, which after enzymatic hydrolysis, can locally stimulate growth of tumors that are estrogen responsive.

Breast Neoplasms↗
PubMed1985

The naturally occurring C-17 fatty acid esters of estradiol are long-acting estrogens.

C-17 fatty acid esters of estradiol are naturally occurring biosynthetic metabolites of estradiol. A representative component of this family of esters, estradiol-17-stearate, was studied in order to determine the estrogenic properties of these unusual hydrophobic steroids. Following the classical estrogen bioassay, a solution of this ester in oil was injected subcutaneously into immature rats once a day for 3 days. There was little effect on the uterus on the first day after the third injection. However, on subsequent days a large stimulation of uterine growth occurred. The course of this estrogenic effect was exactly opposite to that obtained with estradiol. In order to eliminate the possibility that this effect on the time course of estrogenic stimulation was caused by increased solubility of the hydrophobic esters in the carrier oil, the steroids were administered to adult ovariectomized animals in aqueous medium via a single intravenous injection. The uterotrophic response to estradiol was maximal at 12 h and was completely dissipated in 48-60 h. Estradiol-17-stearate produced a uterotrophic effect of twice the duration of estradiol. In the immature rat, aqueous intravenous injections of estradiol-17-stearate produced a greater uterotrophic effect than estradiol and this effect was still maximal 96 h later. In addition, this single injection of estradiol-17-stearate advanced the time of vaginal opening, a marker for puberty in the female rat. The mechanism of the prolonged estrogenic stimulation was investigated by studying the steroidal content of the uterus after injecting [3H]estradiol and [3H]estradiol-17 -stearate i.v. into immature rats. At 1 and 4 h there was significantly more radioactivity in the uteri of the [3H]estradiol treated animals. At later times (8 h and onwards) the total radioactivity in the uterus did not differ appreciably between the two groups. However at these later times, the amount of [3H]estradiol was far greater in the uteri of animals receiving [3H]estradiol-17-stearate. Consequently, the prolonged estrogenic effects of the endogenous C-17 fatty acid esters of estradiol are caused by the increased duration of the estrogenic signal. It is hypothesized that one of the roles of the fatty acid is to protect the steroid nucleus from metabolism and thereby prolong the life of the parent C18 steroid. Thus, the results of these experiments are consistent with the family of endogenous alkyl esters of estradiol having a physiological role as long-acting estrogens.

Animals↗
PubMed1985

The clearance and metabolism of estradiol and estradiol-17-esters in the rat.

The C-17 fatty acid esters of estradiol are naturally occurring estrogens which have been shown to circulate in blood. They are long-acting estrogens, analogous to the synthetic alkyl and aryl esters of estrogens which have been used pharmacologically for decades. To determine the mechanisms involved in the prolonged stimulation evoked by these nonpolar estrogens, several C-17 alkyl esters were synthesized and labeled with 3H at C-17 alpha, and their metabolism and clearance were studied and compared to those of estradiol in rats. The conversion of the C-17-3H to 3H2O was used as a marker of metabolism. While the clearance of the long chain esters from blood is somewhat slower than that of estradiol (t 1/2 = approximately 16 vs. 2 min, respectively), the rates of metabolism are dramatically different. For example, the t 1/2 of metabolism for two representative esters, estradiol-17-stearate and arachidonate, are 580 and 365 min, respectively, while the t 1/2 of metabolism for estradiol is about as fast as its clearance from blood (approximately 2 min). When the effect of chain length was studied, it was found that for the smaller esters, there was an inverse relationship between the size of the acyl group and the clearance from blood, i.e. the longer carboxylic acids were cleared more slowly. However, when the acyl group was lengthened from C12 to C14, the rate of clearance increased and was even faster with C18. Nevertheless, with all of the esters tested, the rate of metabolism steadily decreased as the chain length increased. These results are interpreted as indicating that the control point or rate-limiting step in the metabolism of the estradiol esters is the esterase that hydrolyzes the ester to estradiol. Thus, the prolonged estrogenic action of the C-17-alkyl esters is due to the slow release of estradiol from this hydrophobic reservoir.

Animals↗
PubMed1985

Metabolism of estradiol fatty acid esters in man.

Fatty acid esters of estradiol coupled at the C-17 position are naturally occurring metabolites of estradiol (E2). They have been recovered after incubation of E2 with human tissue and identified in human plasma. We investigated the primary metabolic transformation of E2, namely C-17 oxidation, of two representative fatty acid esters, in five normal subjects (four men and one woman), aged 25-55 yr, using a radiometric method. The transfer of tritium from the C-17 alpha position to tritium water after iv injection of free E2 was compared to that of E2-17 beta-stearate and E2-17 beta-arachidonate. Both esters were oxidized at the C-17 position to a greater extent than was free E2. In addition, the oxidation of the E2 fatty acid esters proceeded more slowly. Thus, the time necessary to reach half the maximal extent of reaction ranged from 30-45 min for the three E2-17 beta-arachidonate studies and from 2.5-4 h for the five E2-17 beta-stearate studies, while that for free E2 was less than 15 min in each instance. The disappearance of intact E2-17 beta-stearate from plasma after bolus injection was studied in two subjects (one of the five above and one additional subject). The t1/2 values calculated for the 0-60 min period were 24 and 16 min. The rate of disappearance E2-17 beta-stearate was slower than that of E2. The biological activity of E2 esters is thought to reside in their ability to be converted, by hydrolysis, to free E2. The E2 esters must undergo hydrolysis before the oxidation of free E2 to estrone can proceed. Since the oxidation reaction is extremely rapid, studying the rate of oxidation after injection of the E2 fatty acid esters provides an index of their in vivo hydrolysis and, thus, a measure of their subsequent biological activity as the free hormone. The unsaturated arachidonate ester of E2 was oxidized at a faster rate than the saturated stearate ester; this implies that the hydrolysis of the arachidonate ester was faster. Evidence of continuing oxidation of E2-17 beta-stearate at times well after its level in plasma markedly decreased indicates that the ester is removed from the circulation before its subsequent hydrolysis and oxidation. We conclude that in man, the C-17 oxidation of both E2-17 beta-arachidonate and E2-17 beta-stearate proceeds more slowly but to a greater extent than that of the free steroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1984

The interaction of C-17 esters of estradiol with the estrogen receptor.

Various C-17 alkyl esters of estradiol (E2) were tested as ligands for the estrogen receptor. The naturally occurring fatty acid esters, represented by E2-17-stearate, E2-17-palmitate, and E2-17-arachidonate, did not compete with [3H]E2 for receptor-binding sites. [3H]E2-17-stearate did not bind in a specific or saturable manner to uterine cytosol. On the other hand, the long-acting pharmacological esters of E2, E2-17-acetate, -propionate, -valerate, etc., were highly effective in competing for the binding of [3H]E2 to the uterine receptor. However, it was found that these short chain esters were hydrolyzed to E2 when incubated with uterine cytosol under the conditions used for the binding assay. Consequently, [3H]E2-17-acetate and [3H] E2-17-valerate were tested as ligands in an assay in which the hydrolytic activity was minimized by partially purifying the receptor with (NH4)2SO4 precipitation and by limiting the duration of the incubation. In this assay there was no specific binding of the C-17-valerate ester. There was a relatively small amount of specifically bound radioactivity in the incubation of the acetate ester, but upon high performance liquid chromatographic analysis, the specifically bound steroid was found to be E2 and not the ester. These results indicate that the C-17 esters of E2, from C2 to the long chain fatty acids, all of which are potent long-acting estrogens, exert their estrogenic effects only after hydrolysis to the free C18 steroid.

Animals↗
PubMed1983

Microsomal 5-ane-3 beta-hydroxysteroid oxidoreductase from pubertal rat Leydig cells: partial purification and characterization.

A 3 beta-hydroxysteroid oxidoreductase which acts on 5 alpha (beta)-reduced C19 and C21 steroids (5-ane-3 beta-hydroxysteroid oxidoreductase; 5-ane-3 beta-HSO) has been solubilized from pubertal rat Leydig cell microsomes and purified 300-fold by ion exchange and gel filtration chromatography. The partially purified enzyme is stable only in the presence of 0.4 M NaCl and appears to exist as a molecule having a molecular weight of 35,000 or as aggregates with a molecular weight in excess of 150,000. NAD+ and NADH+ are used exclusively as cofactors. The velocity of the steroid oxidation reaction was unaffected by either Ca2+ or Mg2+. The steroid oxidation reaction has a pH optimum between 8.0 and 8.5, a temperature optimum at 35 degrees C and an activation energy of 12,850 cal/mol. The pH optimum of the steroid reduction reaction is 6.6. A variety of 5 alpha-reduced C19 and C21 steroids can be utilized as substrates. Treatment of microsomes with phospholipase A2 resulted in a 26 to 90% loss of enzyme activity, paralleling decreased microsomal phospholipid content, and suggesting a role for phospholipids in 5-ane-3 beta-HSO activity. Assays with combined substrates indicate that one enzyme is responsible for activities observed with 5 alpha- and 5 beta-reduced C19- and 5 alpha-reduced C21-3 beta-hydroxysteroids. Purification data indicate that the 5-ane-3 beta-HSO and the 5-ene-3 beta-hydroxysteroid oxidoreductase:isomerase are distinct enzymes.

Animals↗