Leonard R. Axelrod

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Abstract Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals. Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell. Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis. Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.

Reduced high affinity 5α-dihydrotestosterone receptor capacity in the ventral prostate of the aging rat

Abstract Cytoplasmic extracts of ventral prostates obtained from young (50–90 day old) rats 24 hours post orchidectomy contained high affinity binding activity for 5α-dihydrotestosterone which migrated with a sedimentation coefficient of 10–11S on linear sucrose gradients. By contrast, when cytoplasmic extracts were prepared from ventral prostates of aging rats (10–14 months old), 80 percent of the animals evidenced a complete absence of 10–11S binding activity. A single extract prepared from the ventral prostates of the remaining aging animals in the 10–14 month age category had a level of 10–11S binding activity that was about 20 to 25 percent of the value routinely obtained for identical preparations from young animals. Cytoplasmic extracts prepared from the ventral prostate of animals older than 14 months were always found to be devoid of 10–11S binding activity.

Urinary metabolites of 4-14C-progesterone in the baboon (Papio spp.)

Abstract The metabolism of intravenously administered 4- 14 C-progesterone was studied in papio sp. An average of 37% of the radioactivity was recovered in the urine and 15% in the feces. In the urine, 7% of the identified radioactivity was in the free form, 12% as sulfate and 59% as glucuronide. More polar, unidentified material represented 22%. The major, definitively identified metabolites were pregnanediols (41%) and androsterone (36%). Only the 3α,20α-pregnanediol isomers were found; the 5α and 5β forms were present in approximately equal quantities. It was estimated that progesterone production of the baboon corpus luteum proceeds at a much slower rate than in man.

A solid-phase radioimmunoassay for estrogen by estradiol-17β antibody covalently bound to a water insoluble synthetic polymer (enzacryl AA)

Abstract A solid-phase radioimmunoassay for estrogens that has both high accuracy and precision has been developed which uses covalent binding of an antibody to a water insoluble carrier (Enzacryl AA). Several time-consuming and error-producing steps have been eliminated by having in one unit the antibody, the tracer activity and the means for separating the bound and unbound fractions. The antibody for estradiol was produced by the immunization of ewes with estradiol-17β 17-hemisuccinate coupled to bovine serum albumin. Diazotized Enzacryl AA (Koch-Light Laboratories) was used to form the polymer. The percent recovery (accuracy) of the method is 97–103%, while the coefficient of variation (precision) varied from 4–10%. The standard deviation of regression for estrone was ±8.9 picograms (pg) while that of estradiol was ±11.4 pg. The detection limit of the assay, taking into account procedural losses as well as the recovery aliquot, was 75–100 pg.

Color tests for the detection of sterols and estrogens on filter paper

Abstract 1. 1. Six tests have been devised for the detection of the estrogens in microquantities on filter paper. 2. 2. Three of the tests have also been applied to the detection of the sterols. 3. 3. Generally, the sensitivities of the tests range from 2 to 8 μg. of compound/sq. cm. of paper. 4. 4. An array of colors and fluorescences for each compound is helpful for the qualitative separation and identification of the particular estrogen or sterol. 5. 5. Two of the tests are applicable to steroid compounds other than the estrogens and sterols. 6. 6. These tests are applicable to use on paper chromatograms without significant variation in color, fluorescence, or sensitivity.

The metabolism of estrone-16-C14 in bovine blood☆

Abstract 1. 1. Estradiol-17α was found to be the major conversion product of estrone-16-C 14 in pregnant cow blood. 2. 2. Two other phenolic conversion products were found but not identified. 3. 3. The results were discussed in the light of previous investigations.

The extraction of corticosteroids from blood and tissues by dialysis

Abstract A technique is described for the extraction of corticosteroids from blood and tissues by dialysis. The data collected suggest the further possibility of using fractional dialysis as a method for the separation of steroids. The advantages of the new method over those previously available are discussed.

The conversion of 2-hydroxyestradiol-17β to 2-hydroxy and 2-methoxy metabolites in human urine☆

Abstract Intravenous infusions of 1–200 mg. 2-hydroxyestradiol-17β in plasma were administered to four postmenopausal women. The following urinary metabolites were definitively identified: 2-hydroxyestrone, 2-hydroxyestriol, 2-methoxyestrone, 2-methoxyestradiol-17β, and 2-methoxyestriol. The origin of the urinary estrogen methyl ethers appears to be by way of transmethylation of the 2-hydroxylated compound, or as a result of further metabolism of 2-methoxyestrone.

The metabolism of hydrocortisone in the isolated perfused dog liver

Abstract Hydrocortisone was perfused through a male dog liver. Six compounds have been identified on the basis of presumptive evidence: Pregnane-3α, 17α,20α,21-tetrol-11-one, pregnane-3α, 11β,17α,21-tetrol-20-one, pregnane-3α,17α,21-triol-11,20-dione; Δ4-pregnene-11α, 17α, 21-triol-3, 20-dione; Δ4-pregnene-17α,21-diol-3,11,20-trione; and Δ4-androstene-11β-ol-3,17-dione.

The effect of cofactors on steroid biosynthesis in normal ovarian tissue.

Abstract Normal ovarian tissue from cycle day 7 was minced and incubated with isomolar quantities of labelled pregnenolone and progesterone with and without the addition of cofactors (ATP, NAD + , NADH, NADP + NADPH). Only 4–10% of the substrates were metabolized without cofactors, whereas in their presence, the yield rose to 35% of progesterone and 26% of pregnenolone. Cofactors enhanced the activity of 3β-01 dehydrogenase, 17-hydroxylase, 17-ketosteroid reductase, and aromatizing activity, while they did not appear to affect desmolase significantly. 19-Nortestosterone was accumulated in the absence of cofactors.