Precursor role of 15alpha-hydroxyestradiol and 15alpha-hydroxyandrostenedione in the formation of estetrol

Studies were designed to elucidate the origin of estetrol (15alpha-hydroxyestriol (estra-1,3,5(10)triene-3,15alpha,17beta-tetrol) or E4) during late human pregnancy. 3H-Labelled 15alpha-hydroxyestradiol (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17beta-estradiol (estra-1,3,5(10)-triene-3,17beta-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15alpha-hydroxyandrostenedion (15alpha-hydroxyandrost-4-ene-3,17-dione or 15delta4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5--6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)-triene-3,16alpha,17beta-triol or E3) containing 14C only and 15alpha-hydroxyestrone (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15delta4 and only partially via 15E2. When administered to the fetus E2 and 15delta4 contributed approximately equal amounts to urinary E4. The yield of 15alpha-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15alpha-hydroxylase. 15delta4 was a better precursor than E2 for urinary 15E2.     

Preparation of specific antisera to 15alpha-hydroxyestrogen 15-N-acetylglucosaminides.

3-(1-Carboxypropyl) ether derivatives of 15alpha-hydroxyestradiol 15-N-acetylglucosaminide (15alpha-OHE2 15NAG) and 15alpha-hydroxyestriol (E4) 15NAG were synthesized and conjugated with bovine serum albumin. Antisera elicited in rabbits possessed high affinity and specificity for the 15alpha-hydroxyestrogen (15alpha-OHEs) 15NAG, exhibiting no significant cross-reactivity with 15alpha-OHEs and their positional isomers such as 16NAG and 17NAG. Enzyme immunoassay methods developed by using the purified antisera and horseradish peroxidase-labeled antigens were applied to the measurement of 15alpha-OHEs 15NAG and E4 15NAG in normal pregnancy urine. We demonstrated for the first time that the conjugation of N-acetylglucosamine to E4 occurs at the C-15alpha position.     

Preparation of specific antisera to 15alpha-hydroxyestrogens.

The synthesis of haptens of 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol, and 15alpha-hydroxyestriol (estetrol) was undertaken, to obtain specific antisera required for enzyme immunoassay. 3-(1-Carboxypropyl) ethers of these 15alpha-hydroxyestrogens were prepared and conjugated with bovine serum albumin and horseradish peroxidase. The specificity of antisera elicited against bovine serum albumin conjugates was checked by the enzyme immunoassay by using horseradish peroxidase-labeled antigen, and proved to be satisfactory in terms of cross-reactivities to related compounds.     

Isolation fo 15alpha-hydroxypregnenolone and 15alpha-hydroxydehydroisoandrosterone from human pregnancy urine.

15alpha-Hydroxydehydroisoandrosterone and 15alpha-hydroxypregnenolone were isolated from hydrolyzed extracts of human late pregnancy urine and identified by means of the isotope dilution technique. In two separate determinations the excretion rate of 15alpha-hydroxydehydroisoandrosterone was found to be 1.7 and 3.2 microgram per day while that of 15alpha-hydroxypregnenolone was 1.7 and 2.9 microgram per day. It is postulated that 15alpha-hydroxydehydroisoandrosterone might serve as a precursor of 15alpha-hydroxylated estrogens already isolated from late pregnancy urine. Similarly, 15alpha-hydroxypregnenolone might be an endogenous precursor of 15alpha-hydroxyprogesterone.     

Studies on the biosynthesis in vivo and excretion of 16-unsaturated C19 steroids in the boar

1. In one experiment [7alpha-(3)H]pregnenolone was infused continuously for 12min into the left spermatic artery of a sexually mature boar and blood was collected during this period by continuous drainage from the spermatic vein. After infusion, the testis was removed and immediately cooled to -196 degrees C. 2. From both the testicular tissue and the spermatic venous plasma, (3)H-labelled 16-unsaturated C(19) steroids were isolated and characterized and their radiochemical purity was established. 5alpha-Androst-16-en-3alpha- and 3beta-ol occurred mainly as sulphate conjugates and to a lesser extent as free steroids. Only traces of these alcohols occurred as glucosiduronate conjugates. 5alpha-Androst-16-en-3-one was found in the free (ether-extractable) fraction. 3. The isotope concentration of each of the (3)H-labelled 16-unsaturated C(19) steroids in testicular tissue was different from that in spermatic venous plasma. 4. The ratios of tritiated 5alpha-androst-16-en-3alpha- and 3beta-ol (free steroids) to their respective sulphate conjugates in the testicular tissue were less than the ratios of the same compounds in the spermatic venous plasma. The possibility that the sulphates are partially hydrolysed by testicular sulphatases before secretion is discussed. 5. In a second experiment, a continuous close-arterial infusion of [7alpha-(3)H]pregnenolone into the left testis was performed over a 200min period and all the urine that accumulated during the infusion was collected for analysis. 6. No (3)H-labelled 16-unsaturated C(19) steroids were detected in the urine as free steroids. Only a trace of 5alpha-androst-16-en-3alpha-ol was detected conjugated as glucosiduronate, whereas the corresponding 3beta-alcohol occurred mainly as glucosiduronate and to a lesser extent as sulphate. 7. The absence of 5alpha-androst-16-en-3beta-ol glucosiduronate in the spermatic venous blood and its presence in considerable amount in the urine may be attributed to hepatic glucuronyl transferase activity.     

Evidence for 15alpha- and 7alpha-hydroxylase activity in gonadal tissue of the early-life stages of sea lampreys, Petromyzon marinus

Gonads of premetamorphosing larval (PML), transforming (TL) and newly metamorphosed (juvenile) sea lampreys (JL) (Petromyzon marinus) were incubated in vitro with tritiated pregnenolone ([(3)H]P(5)), progesterone ([(3)H]P(4)), and androstenedione ([(3)H]A(4)) to identify the major products of steroidogenesis in early developmental stages. Reverse-phase high-performance liquid chromatography, using two mobile phase gradients, was used to separate the radioactive steroid metabolites. 7alpha-Hydroxylase activity was evident, based on the loss of radioactivity from [(3)H]P(5) labelled at position 7, appearing as tritiated water, and on the appearance of radiolabelled 7alpha-hydroxypregnenolone in the incubation medium. In addition, there was evidence of the synthesis of 15alpha-hydroxylated steroids from the three steroid precursors used. For the progestogen precursors, one of the major 15alpha -hydroxylated metabolites synthesized by both testis and ovarian tissue co-eluted with authentic 15alpha-hydroxyprogesterone, and for [(3)H]A(4), the product was predominantly [(3)H]15alpha-hydroxyandrostenedione. Additional polar steroids were produced, some of which co-eluted with authentic 15alpha-hydroxytestosterone and 15alpha-hydroxyestradiol, whereas others could not be correlated with the authentic 15alpha- or 15beta-hydroxylated steroids available. Ovarian tissues from PML and TL developmental stages synthesized several very non-polar compounds, some of which were present as unconjugated compounds, and others only in the conjugated fraction. These molecules had retention times consistent with pregnanes, and their presence in the incubation medium was therefore indicative of the presence of 5alpha-reductase. These metabolites were not present in the incubation medium from testis, or the JL ovary, suggesting that there is no expression of 5alpha-reductase activity in these tissues. Traces of 17beta-estradiol were found in the incubation medium from ovarian tissue incubated with P(5), but not following incubation with P(4) or A(4). Testosterone was not present in the incubation medium from either ovarian or testis fragments incubated with any of the substrates used.     

Tissue distribution of retinol and its metabolites after administration of double-labelled retinol.

The tissue concentrations and distribution of radioactivity present in retinol and its metabolites were investigated in vitamin A-deficient rats 24h after injection of physiological doses (10mug) of [6, 7-14C2, 11,12-3H2] retinol. The highest concentration of radioactivity was observed in the adrenals, followed by kidney, spleen, liver, intestine and blood. The total radioactivity was greatest in urine, followed in descending order by liver, kidney, blood and intestine. The 14C/3H ratios of crude light-petroleum extracts in the liver, intestines, lungs, heart and faeces were similar to the ratio of the injected retinol dispersion. However, the 14C/3H ratios in the adrenals, kidney, spleen, blood, brain and urine were quite different from that of injected retinol. Alumina chromatography of the kidney and intestinal extracts demonstrated that retinol and retinyl palmitate are the principal forms of vitamin A present. However, alumina chromatography of the liver extract did not reveal the presence of retinol but yielded a major compound with a low 14C/3H ratio. That this compound was not retinol was shown by its inability to react with ethanolic HC1 to yield anhydroretinol. The distribution of radioactivity in ether-soluble, acidic and water-soluble fractions of urine indicated that most of the radioactivity was present in the acidic and water-soluble fractions. The 14C/3H ratios in ether-soluble and acidic fractions were higher than that of injected retinol, whereas in the water-soluble fraction the ratio was similar to the injected material.     

Release of free and conjugated forms of the putative pheromonal steroid 11-oxo-etiocholanolone by reproductively mature male round goby (Neogobius melanostomus Pallas, 1814)

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.     

Metabolic fate of ethynodiol diacetate in the baboon.

The enterohepatic circulation and metabolism of ethynodiol diacetate (3beta,17beta-diacetoxy-17alpha-ethynyl-estr-4-ene) in baboons were studied following the intravenous injection of this contraceptive steroid labeled with 14C (4-position) and with 3H (in either the 3- or 17-acetoxy moieties). Bile and urine from four baboons with biliary fistulas and urine from four intact baboons were collected for 7 hours. On the average, 40% and 44% of the injected dose were excreted in the bile and urine, respectively. Only 48% was recovered in the urine of intact baboons. Analysis of these excretion rates indicates an insignificant enterohepatic circulation of this compound. The steroid was excreted mostly (over 80%) as a glucosiduronate in urine and bile. Very little excretion of the 3-acetoxy compound was detected in the urine or bile at any time interval. 17-Monoacetoxy compounds, however, were detected both in urine and bile, suggesting a difference in the rate of in vivo hydrolysis of the 17beta- vs. the 3beta-acetate.     

Biliary metabolites of testosterone and testosterone glucosiduronate in the rat

A mixture of testosterone-4-¹⁴C and testosterone-1,2-³H-17-glucosiduronate was intraperitoneally administered into male and female rats with bile fistulas. Biliary metabolites were separated and purififd by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the blue. 5β-Androstane-3α, 17β-diol was found principally in monoglucosiduronate fraction and was produced preferentially from the injected conjugate in both sexes. Very marked sex differences from the injected conjugate in both sexes. Very marked sex differences were observed in the following metabolites: Androsterone was present only in the female as monoglucosidironate, which was preferentially derived from testosterone. 5α-Androstane-3α,17β-diol was identified in both monoglucosiduronate and diconjugate fractions of the female, which was formed significanrly more from the conjugate than testosterone. These findings provide evidence that testosterone glucosiduronate could be converted directly into 5α-steroids as well as 5β-ones $\text{in}$$\text{vivo}$. In marked contrast, the major portion of testosterone was metabolized to polar steroids in the male.     

Improved synthesis of 16alpha-hydroxylated androgens: intermediates of estriol formation in pregnancy.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.     

In vivo metabolism of 3-ketoceramide in rat brain

Eight hours after intracerebral injection of a double-labeled 3-ketoceramide⁴, [1-¹⁴C]lignoceroyl 3-keto [1-³H]sphingosine, various brain sphingolipids were isolated. Free ceramide and the ceramide portions of nonhydroxy cerebroside and sphingomyelin were further fractionated into subgroups containing longer-chain or shorter-chain fatty acids. Nonhydroxy ceramide, nonhydroxy cerebroside and sphingomyelin containing longer-chain fatty acids had significant quantities of radioactivity with ³H/¹⁴C ratios similar to each other but lower than that of the injected material. The sphingolipids containing shorter-chain fatty acids were also significantly labeled; however, the ³H/¹⁴C ratios were much higher than that of the injected material. Hydroxy-ceramide and sulfatides contained very little radioactivity. However, hydroxy-cerebroside contained an amount of radioactivity comparable to that of the longer-chain nonhydroxy cerebroside with a similar ³H/¹⁴C ratio. It is proposed that the injected 3-ketoceramide was converted into ceramide, cerebroside, and sphingomyelin and that the fatty acids of these lipids were partly replaced by other fatty acids during the metabolic conversions.     

The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.     

Biosynthesis of carnitine and 4-N-trimethylaminobutyrate from 6-N-trimethyl-lysine

The conversion of 6-N-[Me-(14)C]trimethyl-lysine into carnitine and 4-N-trimethylaminobutyrate (butyrobetaine) was demonstrated in rats kept on a lysine-deficient diet. After the rats were given [(14)C]trimethyl-lysine for 4 days, a total of 17% of the injected label was recovered as carnitine from carcass and urine extracts. Another 8% of the trimethyl-lysine label was converted into 4-N-trimethylaminobutyrate, most of which was recovered from the urine. The conversion of trimethyl-lysine into the above two metabolites supports the pathway of carnitine biosynthesis as lysine+methionine --> 6-N-trimethyl-lysine --> 4-N-trimethylaminobutyrate --> carnitine. In addition, three other metabolites representing 2% of the injected dose were recovered. Only an insignificant portion of the label was recovered as free trimethyl-lysine from the carcass, whereas 22% of the injected label was recovered in the urine. A relatively low specific radioactivity in carnitine was found when 5-N-[Me-(14)C]trimethylaminopentanoate and 6-N-[Me-(14)C]trimethylaminohexanoate were administered to rats in amounts similar to the [(14)C]trimethyl-lysine, suggesting that they were not free intermediates.     

Androgen metabolism in sheep

³H-Testosterone (³H-T) plus ¹⁴C-androst-4-ene-3.17-dione (A-dione) and ³H-epi-testosterone (17α-hydroxy-4-androsten-3-one) (epiT) plus ¹⁴C-T were injected intravenously into two male sheep with bile fistulae, respectively. Urine and bile samples were collected at intervals for 4–8 hours and analyzed by the use of DEAE-Sephadex A-25 and Lipidex 5000 columns, TLC, and paper chromatography; the aglycones were identified by co-crystallization with authentic standards.Five fractions were obtained from urine and bile: unconjugated, glucosiduronates, sulfates, sulfo-glucosiduronates and disulfates. In urine, the major conjugates were glucosiduronates, while sulfates predominated in bile. About 80–90% of recovered radioactivity was found to be either glucosiduronates or sulfates. Among the metabolites identified, epi-T was the principal one, accounting for 10–15% of the administered doses. Conversion to 17α-hydroxysteroids thus appears to be a major route of metabolism of the androgens administered in sheep. Other metabolites in the glucosiduronate and sulfate fractions were androsterone, etiocholanolone (3α-hydroxy-5β-androstan-17-one), 5β-androstane-3α, 17β-diol, two unknown diols and polar metabolites. The results indicated that androgen metabolism is somewhat unusual in sheep, as compared with other animals and the human.     

Synthesis of new analogs of 15-ketosterine from ergosterine

Ergosteryl acetate was converted through three stages into 3 beta-acetoxy-24-methyl-5 alpha-cholesta-8(14),22-diene-15-one in 32% overall yield. The product was transformed to 3 beta-hydroxy-24- methyl-5 alpha-cholesta-8(14),22-diene-15-one, 3 alpha-hydroxy-24-methyl-5 alpha-cholesta-8(14),22-diene-15-one, and 24-methyl-5 alpha-cholesta-8(14),22-diene-3,15-dione. The compounds were characterized by 1H and 13C NMR spectra. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.     

The role of cytosolic proteins in the intracellular transport of haem in rat liver. A dual-label approach.

After consecutive injections of delta-amino[3H]- and -[14C]-laevulinic acid, the incorporation of the two labels into haem associated with different subfractions of the liver was determined. Marked differences in the 14C/3H ratios were observed between haem associated loosely and tightly with microsomes and mitochondria and haem associated with three subfractions of the cytosol obtained by gel filtration. The effect of changing the amounts of delta-aminolaevulinic acid injected and of changing the interval between injections and killing of the animal on the ratios of labels in the haem of each subfraction was studied. The results are discussed in terms of the flow of haem from the mitochondria to other parts of the cell via putative cytosolic carrier proteins.     

Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi

The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.     

Estradiol-15 alpha-hydroxylation: a new avenue of estrogen metabolism in peri-implantation pig blastocysts

Pig blastocysts have the capacity to convert estradiol into catechol estrogens. Our present study shows that they also have the capacity to hydroxylate estradiol in cycloaliphatic C-atom 15, and this aliphatic hydroxylation reaction is more predominate than the aromatic hydroxylations. The conversion of [4-14C]estradiol to [4-14C]15 alpha-hydroxyestradiol by mitochondrial-rich/microsomal fractions was examined by isolation of this product using reversed phase high-performance liquid chromatography (HPLC) attached to a radiometric flow detector, and its identification by gas chromatography-mass spectrometry. The enzyme kinetics for estrogen 15 alpha-hydroxylase were performed in the pig blastocyst obtained on Day 13 of pregnancy (Day 0 = first acceptance of the male). The enzyme follows classical Michaelis-Menten kinetics. The apparent Kms for estradiol were 2.47 and 1.85 microM, and the apparent Vmaxs were 0.25 and 0.197 nmol/mg/min in the mitochondrial-rich and microsomal fractions, respectively. The enzyme activity was inhibited by different steroidal compounds and non-steroidal estrogens, as well as by CO, SKF-525A, piperonyl butoxide and antibody to cytochrome P450 reductase. Ontogenesis of the blastocyst's estrogen 15 alpha-hydroxylase follows a similar pattern to that of estrogen-2/4-hydroxylase. Thus, highest activity was observed on Days 12 and 13 and lowest was on Day 15 of pregnancy. Furthermore, the enzyme is abundant primarily in the extraembryonic tissues rather than in the embryo proper. The abundance of the enzyme in the extraembryonic tissues, and its surge at a critical time of pregnancy recognition and just prior to implantation suggest that 15 alpha-hydroxylated estradiol could be involved in these processes.