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Y Osawa

Publications and source records attributed to Y Osawa.

At least 199 records · Page 11Linked to original sources
PubMed1977

Localization of estrogen synthetase in the chorionic villus fraction after homogenization of human term placenta.

In previous investigations of human placental estrogen synthetase (aromatase), activity was found in the microsomal and mitochondrial fractions after homogenization and subcellular fractionation of placental tissue in isotonic sucrose. We report here that the majority of the placental aromatase sediments in a much heavier particulate fraction than the microsomal or mitochondrial fractions. After low speed centrifugation of the placental homogenate, only about 20% of the homogenate aromatase activity was measured in the supernatant, the precursor of the microsomal and mitochondrial fractions. Even after extensive homogenization in high speed shearing-type blenders and tight-fitting glass homogenizers the majority of the placental aromatase (more than 70%) consistently sedimented at very low centrifugal forces (100 x g ro 10 min) independent of whether the suspending medium contained up to 1.2M sucrose, or IM NaCl, or 50% glycerol. By selectively separating components of the low speed pellet via a sieving procedure or manual dissection of placental tissue, we found that the majority of the enzyme activity is associated with placental chorionic villus fragments. Several extensively washed preparations of villus fragments with aromatase specific activities similar to that found in placental microsomes were synthesis in the villus aromatase fraction was determined to be 1beta,2beta-H removal which is identical with that by placental microsomes and human ovaries. Evidence for the presence of cytochrome P-450 in the villus fraction was obtained by measuring the carbon monoxide-dithionite difference spectrum.

Androstenedione↗
PubMed1976

Active-site-directed inactivation of aromatase from human placental microsomes by brominated androgen derivatives.

Several brominated androgen derivatives were tested for their ability to inactivate microsomal aromatase from term human placenta. In the experimental protocol, the microsomal homogenate was incubated either with androstenedione or a brominated derivative of androstenedione (16alpha-bromo-6-ketoandrostenedione, 16alpha-bromoandrostenedione, 7alpha-(3'-bromoacetoxypropyl)androstenedione, 6alpha-bromoandrostenedione, or 6beta-bromoandrostenedione) and reduced nicotinamide adenine dinucleotide phosphate in a nitrogen saturated buffer composed of glycerol, ethylenediaminetetraacetic acid, and dithiothreitol in tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4) under nitrogen at 4 degrees C with shaking. After the incubation period, the microsomes were recovered by centrifugation and washed once before determining aromatase specific activity. The brominated androgen derivatives which inactivated aromatase were 7alpha-(3'-bromoacetoxypropyl)androstenedione and 6alpha-bromoandrostenedione. The structures of 6alpha- and 6beta-bromoandrostenedione were unequivocally established by single crystal x-ray diffraction techniques. The extent of the enzyme inactivation by 6alpha-bromoandrostenedione was linearly proportional to the logarithm of its concentration. The evidence that this inactivation occurs at the aromatase active site is that androstenedione, when coincubated with 6alpha-bromoandrostenedione, protected aromatase from this inactivation. Progesterone provided much less protection than androstenedione. Furthermore, both 6alpha- and 6beta-bromoandrostenedione are competitive inhibitors of androstenedione aromatization, as determined by a Lineweaver-Burk plot, and 6alpha-bromoandrostenedione gives the same type I cytochrome P-450 binding spectrum with placental microsomes as androstenedione. These data suggest that 6alpha-bromandrostenedione is effective as an active-site-directed inhibitor of placental microsomal aromatase.

Androstenedione↗
PubMed1976

The metabolic fate of chlormadinone acetate in the baboon.

The metabolic fate of chlormadinone acetate (17alpha-acetoxy-6-chloro-4, 6-pregnadiene-3, 20-dione; CAP) was studied in intact and biliary fistula baboons. The steroid was labeled with 3H at position 1 and with 14C at the carboxyl moiety of the 17alpha-acetate, thus affording the opportunity to ascertain the loss of the 17alpha-acetoxy group and the fate of both labels. The averages of the radioactivity excreted, given as percentages of the amounts injected, and the standard deviations were as follows: In the urine of intact animals after 6 hours, 5.7 +/- 0.2% and 5.5 +/- 0.7% of the 3H and 14C were recovered, respectively. After 6 days, there was 17.5% of the 3H and 16.2% of the 14C in the urine plus 15.3% of the 3H and 16.4% of the 14C in the feces. In baboons with biliary fistulas, the total radioactivity excreted was 7.8 +/- 0.7% of the 3H and 11.6% of the 14C in the urine, and 30.9 +/- 4.4% of the 3H and 30.7% of the 14C in the bile after 6 hours. Glucosiduronates were the predominant conjugates in the urine and bile. The similarity in the urinary excretion of radioactivity in the first 6 hours in intact and biliary fistula animals, the relatively low excretion of radioactivity in the bile and after 6 days in the urine, and the low fecal excretion suggest that the metabolites of CAP are not involved in an extensive enterohepatic circulation in the baboon. Deacetylation of the 17alpha-acetate in CAP was detected in the early collection periods of the urine and bile and constituted a very small percentage of the injected compound. No significant oxygenation of CAP at position 1 was detected. The metabolism of CAP is discussed and compared to our previously reported data on the metabolism of progesterone, ethynodiol diacetate and medroxyprogesterone acetate and the data on other progestogens reported in the literature. It appears that the excretion of CAP is significantly slower in the baboon than that of the other progestogens. The amounts of glucosiduronates of CAP and/or its metabolites formed in vivo are less than those formed with the other progestogens. Also, the extent of deacetylation of the 17alpha-acetate of CAP is much less than that of the 3beta-acetate of ethynodiol diacetate.

Animals↗
PubMed1976

Conformational influence of a 19-methyl substituent in 19-oxygenated steroid structures.

The crystal and molecular structure of (19R)-19-methyl-5-androstene-3beta, 17beta, 19-triol (C20H32O3) has been determined. The crystals are orthorhombic and the space group is P212121. The unit cell parameters are a =11.179 A, b = 21.485 A, and c = 7.328 A. The structure was solved using the direct methods program MULTAN and refined anisotorpically to an R of 7.2% for all data. The methyl substituent on C(19) is located over the B ring and the hydroxyl between the A and C rings. The flexible B ring has a distorted half-chair conformation. The 19R configuration suggests that the reaction mechanism for the formation of this compound proposed by Wicha and Caspi is incorrect. Furthermore, these results indicate that the stereochemical assignment of C(19) by Skinner and Akhtar resulting from a tritiated sodium borohydride reduction is also suspect.

Androstenols↗
PubMed1976

In vivo and in vitro conjugation and metabolism of estrogens by the baboon kidney.

Labeled estrogen was injected into one of the renal arteries of baboons and urine was collected separately from each ureter via ureterostomy at various time intervals over a period of 7 h; the urinary metabolites were analyzed by DEAE-Sephadex chromatography and enzyme hydrolyses. Identification of the aglycones was made by TLC and co-crystallization upon admixture with authentic compounds. When [3H]estradiol-17beta (E2) was injected into one of the renal arteries, the major metabolites were [3H]E2-3-glucosiduronate (E2-3G) and [3H]estrone-3-glucosiduronate (E1-3G); [3H]E2-3G was excreted predominantly into the urine from the injected side during the initial 30 min after injection. Formation of [3H]E1-3G was not detectable for the first 5 min in the urine from either side. However, its excretion gradually increased and the amount was almost equal from both sides in the later hours of collection. Injection of [3H]E1 revealed ready conjugation (as E1-3G) by the kidney without significant reduction at position-17. When E2-3G, labeled with 3H at positions 6,7 and with 14C in the glucuronic moiety, was injected, it was quickly excreted in the urine of the injected side. The ratio of 3H/14C of E2-3G excreted in the urine was the same as that of the injected E2-3G. In the later periods, E1-3G, which had a slightly increased 3H/14C ratio, appeared in the urine. These results indicate that E2 and E1 are conjugated directly in the kidney to form the 3G which is excreted into the urine and that effective dehydrogenation or reduction of the steroids in vivo does not take place in the kidney. The results also show that conjugated E2 (E2-3G) is quickly excreted by the kidney of the baboon without any change in its form and that the E2-3G is dehydrogenated to E1-3G during its general circulation without significant prior removal of the glucuronic moiety. When [3H]estriol (E3) was injected, facile glucuronidation to E3-16G by the kidney was shown. Injection of an equimolar mixture of [14C]E3 and [3H]E2 showed rapid excretion of [14C]E3-16G as opposed to the slower excretion of [3H]E2-3G. These results suggested that the active sites of these conjugating enzyme systems in the kidney may be different.

Animals↗
PubMed1976

Studies of phenolic steroids in human subjects. XIX. Renal conjugation of 15alpha-hydroxyestradiol-3-sulfate.

In order to elucidate further the role of the kidney in the conjugation and excretion of 15alpha-hydroxy-estrogens, we synthesized doubly and singly labeled 15alpha-hydroxyestradiol-3-sulfate (15alpha-OHE-S) and determined its urinary excretory pattern after administering differently labeled 15alpha-OHE2-S into the renal artery and concomitantly into a peripheral vein and by the infusion of doubly labeled 3H-15alpha-OHE2-S-35S into the renal artery. The excretion of radioactivity in the early urinary samples and the conjugate pattern in these urines were determined. About twice as much radioactivity was excreted in the early urine samples originating from the renal artery injection than from samples obtained following peripheral administration, indicating renal conjugation and excretion of the 15alpha-OHE2-S. The latter was preponderantly excreted as the S-NAG with only a small part (about 10%) of the administered sulfate being hydrolyzed to the aglycone. The latter was then either conjugated with N-acetylglucosamine (NAG) or glucuronide, probably also in the kidney. Most of the 15-alpha-OHE2-S could not be accounted for, possibly because of biliary excretion and enterohepatic circulation. The results obtained and those already published indicate that the human kidney is capable of conjugating 15alpha-OHE2, its NAG, and its sulfate conjugates.

Adult↗
PubMed1976

Studies on phenolic studies in human subjects. XX. In vivo conjugation and metabolism of estradiol-17beta in the human kidney.

Labeled estradiol-17beta (E2) was injected into one of the renal arteries of two human subjects. At the same time, an equimolar amount of differently labeled E2 was injected into a peripheral vein. The urinary metabolites were analyzed by DEAE-Sephadex A-25 column chromatography, countercurrent distribution (CCD) and enzyme hydrolyses. Identification was made by statistical analysis of data from CCD, thin layer chromatography (TLC) and co-crystallization upon admixture with authentic compounds. The major urinary metabolites were E2-17glucosiduronate (E2-17G), E2-3G and estriol-16G (E3-16G). The E2-17G was excreted immediately following injection of 14C-E2 into the renal artery of subject no. 1, at a rate which decreased gradually with time; whereas 3H-E2-17G did not appear in the urine until 5 min after injection of 3H-E2 into a peripheral vein. The excretion of 14C-E2-17G was very prominent as opposed to that of 3H-E2-17G; however, the excretion of both 14C- and 3H-E2-17G terminated within 30 min. 14C-E2-3G was excreted immediately following injection, whereas 3H-E2-3G did not appear until 5 min after the injection. Also, the excretion of 14C-E2-3G was more prominent as opposed to that of 3H-E2-3G. The excretion of these compounds was rapid in the initial 15 min after injection and then continued slowly for 1 h. On the other hand, 14C- and 3H-E2-16G appeared at 30 min after injection and the 3H/14C ratio was almost the same as that of the injected compounds. When subject no.2 was injected with the labeles reversed, the results were very similar to those described above. The results indicate that E2 is conjugated directly in the human kidney to form the 17G and 3G and excreted into the urine, whereas the conversion of E2 to E3 occurs systematically rather than in the kidney. In contrast of E3, the kidney appears to play a minor role (no more than 10% of the total E2 is involved) in the conjugation and/or metabolism of E2 in the human.

Estradiol↗
PubMed1976

Improved assay for urinary 17-hydroxycorticosteroids.

We describe a simple, rapid, reliable method for determining urinary 17-hydroxycorticosteroids. A neutral resin (Amberlite XAD-2), which is a non-ionic cross-linked polystyrene, is used to extract and concentrate the steroids, which are then quantitatively determined with the Porter-Silber reaction. Use of the resin eliminates the need for enzymatic hydrolysis and n-butanol extractions, thereby decreasing analysis time considerably; results can be obtained within 3 h of receipt of specimens. Most of the nonsteroidal Porter-Silber chromogens are removed, resulting in a method that is highly specific and sensitive, even at low concentrations (0.6 mg/liter), and so more accurate and reproducible than currently used methods.

17-Hydroxycorticosteroids↗
PubMed1975

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.

Animals↗