THE JOURNAL OF Bmmcmar, CH~ISTRY Vol. 241, No. 1, Issue of January 10, 1966

Printed in U.S.A.

Studies on Testosterone Metabolism

III. THE SELECTIVE “j/3-METABOLISM” OF TESTOSTERONE GLUCURONIDE*

(Received for publication, August 6, 1964)

PAUL ROBEL, ROMIZO EMILIOZZI, AND ETIENNE-EMILE BAULIEU

From the Unit& de Recherches (Institut National de la XantS et de la Recherche MWicale) SW le Mt%abolisme Mo&xlaire et la Physio-Pathologie des St&oides, Laboratoire de Chimie Biologique, Facultd de Mkdecine, Paris 6, France

SUMMARY

Testosterone glucuronide (unlabeled and labeled) was synthesized and injected into three normal men, one preg- nant woman, and one child.

In addition to testosterone glucuronide itself, several urinary metabolites were isolated. Only 5&androstane steroids were obtained. More 5/!Landrostane-3or, 17/3-diol than Sfl-androsterone was found. Meanwhile, a testosterone tracer gave Sa-metabolites, and more S/3-androsterone than S/3-androstan-3a!,l7fi-diol.

These findings provide evidence for a further metabolism of testosterone glucuronide through a selective S&metabo- lism. A direct metabolism of the conjugate giving 5/?- metabolites could be postulated; a metabolic cleavage of testosterone glucuronide seems unlikely, unless it occurred in an undefined organ in which testosterone does not give Sa-compounds. In any case, the glucuronic acid moiety greatly changes the metabolism of testosterone.

Calculations made from the specific activity of urinary testosterone after injection of radioactive testosterone and testosterone glucuronide show a production of approximately 5 % of testosterone glucuronide from testosterone in the body.

During these studies, the structure of urinary testosterone glucuronide has been observed to be mostly 17fl-yl-p-~- glucopyranuronoside, less than 10% (if any) of urinary testosterone glucuronide being possibly 3-enol or diglu- curonide or both.

Testosterone appears in urine, mainly as a glucuronidel (3, 4)

* Part of the data included in this work has been reported at the 1964 Endocrine Society Meeting, San Francisco (Abstract 229), and in a preliminary note (Compt. rend. Acad. Sci., 268, 1331 1964). For the previous papers of this series see References 1 and 2. This work has been supported by the Institut National de la Sante? et de la Recherche Medicale of France, by Contract 63-903 of the Delegation B la Recherche Scientifique et Technique, and by a grant from the Population Council.

1 The trivial names of the steroids and compounds used are: glucuronide, p-n-glucopyranuronoside; 5P-androsterone, 3~ hydroxy-5&androstan-17-one; androsterone, 3a-hydroxy-5a-an- drostan-17-one; epiandrosterone, 3&hydroxy-5a-androstan-17- one; sulfate, ester sulfate.

in yields of approximately 0.2 to 1% of produced (or injected) testosterone. Testosterone glucuronide is formed in vitro from testosterone by the liver (5), and the conjugation is believed to occur at the 17/3-hydroxyl group since the ultraviolet absorption spectrum is characteristic of a conjugated ketone (6). p-Glu- curonidase hydrolysis is used in the measurement of urinary testosterone (4), providing evidence for the ,&glucuronoside struc- ture of the urinary product. The latter result does not a.llow for a distinction between a probable conjugation at the C-17 position of testosterone, a less likely conjugation at the C-3 position of the enol form, or conjugation at both the 17-hydroxyl and the 3-enol sites. Knowledge of the structure of endogenous urinary testos- terone glucuronide is necessary in order to determine its produc- tion rate and its metabolism.

Solubility in water, low plasma concentration (7), elevated renal clearance (7, 8), and t.otal excretion without metabolic transformation (9) have led to the consideration of the steroid glucuronides as typical waste products of the corresponding free steroids that result from detoxication reactions. However, since testosterone glucuronide is a conjugate of an active hormone and not of an inactivated metabolite, it could be an important factor in the transport of testosterone (10). Moreoever, the plasma level of testosterone glucuronide seems to be of the same magni- tude as that of testosterone (11).

The present chemical studies on urinary testosterone glucuro- nide show that the C-17 glucuronide is the most important, if not the unique form of testosterone glucuronide. Testosterone glu- curonide, testosteroneJ4C glucuronide, and testosteroneJH glucuronide were synthesized and injected into human beings. When administered as the glucuronide, testosterone did not yield any 5ol-metabolite. In the urine only, 5&androstane steroids were found. Although injected testosterone glucuronide does not appear to release any free testosterone into the blood stream, it does undergo metabolic conversions. The implications of this metabolism will be discussed. The rate of production of testos- terone glucuronide from testosterone and the possibility of a different origin of testosterone glucuronide will also be discussed.

EXPERIMENTAL PROCEDURE

Subjects (Table I)

Radioactive testosterone glucuronide and testosterone in a few milliliters of 10% ethanol were injected intravenously into normal young men (Experiments 1, 2, and 3) at 9 a.m. The urine was

20

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collected for 4 days after the injection and then pooled. Refer- ence will occasionally be made to a fourth experiment performed by Dr. F. Dray in a normal pregnant woman (9th month).

The urine of a leukemic 8-year-old boy (Experiment 5) was collected for 2 days before and for 4 days after the intramuscular injection of testosterone glucuronide and testosterone-l% dis- solved in 3 ml of 1,2-propanediol.

Administered Compounds (Table I)

Testosterone-i ,%3H-Testosterone-l ,2-3H (Tracerlab) (spe- cific activity, 150 PC per kg) was used in Experiments 1 and 2, and for the preparation of testost.erone-1,2-3H glucuronide used in Experiment 3. The “metabolic stability” of tritium, as far as the formation of 17-ketosteroids and various androstanediols is concerned, was established previously (2).

Testosterone-4-14C-Testosterone-4-14C was synthesized by Dr. H. Flochz as testosterone-4-14C acetate, and then deacetylated by saponification with potassium hydroxide. The testosterone-4- 1% was purified by gradient chromatography on alumina and partition chromatography on Celite. The specific activity was found to be 28 PC per pmole, based on the absorption at 240 rnp. It was found to be more than 95% pure by paper chromatography and by the isotopic dilution technique with the use of pure testos- terone3 as a carrier. This testosterone-4-14C was injected in Ex- periments 3 and 4 and used for the preparation of testosterone-4- 14C glucuronide (Experiments 1 and 2).

Testosterone Glucuronide-The method of synthesis (12) fol- lowed the procedure of Koenig and Knorr (13) already used by Shapiro (14) and Wotiz et al. (15). Essentially, this consists of condensation of test,osterone with methyl la-n-bromo-l-deoxy- 2,3,4-triacetylglucopyranosyluronate in the presence of silver carbonate.

A portion (0.87 g) of testosterone (m.p. 154”, [(Y]:’ = +108” (c = 1% ethanol)) and 1.50 g of methyl lcr-n-bromo-l-deoxy- 2,3,4-triacetylglucopyranosyluronate (m.p. 83%85”, after solidi- fication 105-107”, [a]:’ = +198” (c = 1% chloroform)) (16) were dissolved in 30 ml of anhydrous benzene in a flask which could be attached to a rotating evaporator. Then 0.65 g of silver carbonate (made on the previous day) was added. The flask was connected to the evaporator, rotated for 2 hours in a water bath at 60”, and the benzene was then evaporated in a vacuum. The addition of benzene, the 2-hour rotation at 60”, and the evaporation were repeated twice the same day. The final products were dissolved in benzene and transferred to a silica gel (Davison, 100 to 200 mesh) column (60 g), 30 cm high with a diameter of 2.5 cm. A Celite layer 2 cm high was placed at the top of the column, in order to retain the colloidal silver salts. After a benzene washing, the product (800 mg) was eluted by a benzene-ether (3 : 1) mixture. Two crystallizations from 70% aqueous methanol gave 426 mg of the testosterone glucuronide triacetate methyl ester (3-ketoandrost-4-en-17@yl 2,3,4-triacetylglucopyranosiduronic acid, methyl ester), m.p. 186-188”, pure by thin layer chromatography, [(Y]~” = +20” (c, 1% dioxane). The yield at this stage was 23%. The prod- uct was then dissolved in a mixture of 20 ml of methanol and 5 ml of 1 N sodium hydroxide. The solution was heated to 40” and then left for 48 hours at room temperature. The methanol

2 Testosterone-4-l% is a compound made at the Commissariat B 1’Energie Atomique, France, 1962, 2218.

3 Authentic reference compounds are provided by Roussel- UCLAF, Paris, France.

TABLE I Administration of testosterone and testosterone glucuronide

Experiment

1. Normal man. 2. Normal man. . 3. Normal man. 4. Normal pregnant

woman. 5. Leukemic boy. _.

Injected testosterone

PC K?

3H 2.4 0.02 3H 7.6 0.05 W 9.2 120

1% 0.7 9

_-

-

Injected testosterone glucuronide

PC 14C 2.5 w 0.2 3H 3.5

w 0.5 Unlabeled

nzga 2.2 0.2 0.1

0.4 56

(L In testosterone equivalent.

was evaporated under reduced pressure and the residue was dissolved in 100 ml of distilled water. The water solution was extracted twice with 50 ml of benzene, and then it was evapo- rated to dryness. Crystallization of the residue from 6 ml of water gave 300 mg (m.p. -300”) ; two further crystallixations from water left 80 mg of a compound with m.p. 295-300”, [LY]~:; = +14” + 4”, (c, 1% water), [ar]iii = +24” f 4” (c, 1% water).

testosterone glucuronide, 3H?O (540.54)

C2bH3508Na13H20

Calculated: C 55.54, H 7.64, Na 4.25 Found : C 55.68, H 7.84, Na 4.43

The compound shows a strong absorption at 240 rnp (meth- anol), migrates as a single homogeneous band in the B 20-l system (17) with the same mobility as a sample obtained from Dr. H. Wotiz (15), and it is totally hydrolyzed by &glucuron- idase (Ketodase).

Testosterone-&W Glucuronide and Testosterone-l , Z-3H Glu- curon&-The same procedure used for testosterone glucuronide was followed. Purification of the labeled testosterone glucuron- ide was achieved by Celite and paper partition chromatography. Crystallization of the sodium salt from methanol-water after addition of unlabeled testosterone glucuronide to an aliquot gave a specific activity equal to the theoretical calculated specific ac- tivity. p-Glucuronidase hydrolysis with Ketodase provided additional proof of the structure since the recovery of labeled testosterone was complete.

URINE ANALYSES (FIG. 1)

Hydrolyses, Extractions, and Preliminary Purifications

After the injections, the urine samples were stored in a re- frigerator until they were processed. Mercurithiosalicylic acid was used as a preservative.

In each case, the urine was treated as described previously (18) in order to obtain separately and quantitatively the steroids ex- creted as sulfates and those conjugated with glucuronic acid. The former were obtained by solvolysis (19) and the latter by subsequent enzymatic hydrolysis. Steroids referred to as “glu- curonide” or “sulfate” are those excreted in the urine in the corresponding form, whose analysis was performed after the ap- propriate hydrolysis. It is recognized that the sulfate fraction may contain free steroids, but in previous experiments there was so little radioactivity in the free steroids that this could not ap- preciably affect the results. In Experiment 2, a preliminary dichloromethane extraction of the lst-day urine recovered only an insignificant amount of radioactivity ( < 0.1 yO).

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22 Studies on Testosterone Metabolism. III

I’rine

Vol. 241, No. 1

(;lucuronide fraction I

Sulfate fraction

Ketonic Sonketonio Ketonic

(iradient partit;ion (System E) Alumina 67; (System B) Gradient parrit’;on (System E)

I I ’ Alumina 496 z 47 G

I Alumina 475 Partition Partition Alumina 4%

7 Alumma 491, (System A) (System A) (Syst.em A)’ (System D) (Syst,enl D) (System A) (System A)

I I I I I 71 5p-Androster- Androsterone Testosterone 5p-Androstane- Androstane- Epiandrosterone B&Androsterone Androsterone

one 3a, 17p-diol 3a, 17B-diol

FIG. 1. Procedure of isolation of urinary steroids in Experiments 1 and 2

The steroids obtained from both the sulfate and the glu- curonide fractions were separated into ketonic and nonket,onic fractions with 6he Girard T reagent.

Chromatography

The techniques used were the following.

Free Steroids

System A-Alumina column chromatography, with a gradient elution according to Lakshmanan and Lieberman (20), was done for ketosteroids.

System B-This included a modification of the former for the nonketonic steroids (1).

System C -Silica gel thin layer chromatography was carried out in the dichloroethane-benzene (1: 1)” system (1).

System D-Part,ition column chromatography on Celite 535 column was done according to the method of C. Pidacks as de- scribed by Siiteri (21). Specific systems are described under “Results.”

System E- -Gradient elution partition column chromatography was carried out with ethylene glycol as the stationary phase (22) ; the mixing chamber cylinder contained isooct,ane, and the donor cylinder ethyl acetate.

System F---This consisted of paper chromatography in the ligroin-propanediol syst.em for 17-ketosteroids and testost,erone (23).

System G- Paper chromatography in the cyclohexane-benzene (1: 1) system was done for nonketonic steroids.

Conjugated Steroids

System H-Silica gel thin layer chromatography in the et.her- 1,2-dichloroethane (1:2) system was used for purifying triacetyl glucuronide methyl esters.

System 1- -Florisil adsorption column chromatography (17) was used to separate the free, sulfate, and glucuronide steroids.

4 Proportions by volume for all systems.

System J-Celite 535 partition column chromatography was done with the following systems used by Siiteri (21) for C19- steroid glucuronides: (a) isoortane-tert-butyl alcohol-acetic acid- water (200:550: 125:500);4 (b) isooctane-tert-butyl alcohol-l M ammonia (100 : 300 : 250).

System &-Linear gradient partition chromatography on Celit,e column included several phases: stationary phase, acet.ic acid-water (65 : 250) ; first mobile phase, isooctane-tert-butyl al- cohol (200 : 200) ; second mobile phase, isooctane-terD-butyl alco- hol (40 : 200) (steroid glucuronides) .

System L-Linear gradient partition chromatography was car- ried out on <‘elite column: stationary phase, acetic acid-water (20: 180); first mobile phase, isooctane-et,hyl acetate (100: 100); second mobile phase, ethyl acetate 200 (steroid glucuronides).

System JI- -This paper chromatography for steroid glucuro- nides was the B 20-l system (17).

Detection and Characterization

The Zimmermann reaction for 17-ket,osteroids and t’estos- t,erone, absorption for testosterone at 240 mp, and the SbClr fluorescence chromogen for t,he nonketonic compounds were used for purposes of detection and occasionally for measurement,. Infrared spectrophotometry with a double beam spectrometer (Perkin-Elmer, model 13), wit.h the use of t.he KBr pellet tech- nique, was performed for ident,ification of 5&androsterone. Isotope dilution with pure unlabeled c*ompounds3 was performed in most, experiments, in order to confirm the authenticity and homogeneity of the isolated compounds.

Counting and Calculation

The scintillation count,ing procedure has been described (1). When testosteroneJ4C glucuronide or testosteroneXI glu- curonide was counted, 0.2 or 0.5 ml of methanol was added to the 5 ml of scintillation toluene solution, and the results were corrected for quenching. ,4 similar correcation was made when relatively large amounts of 5a- or 5&androstan-3a, 17/3-diols

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Issue of January lo,1966 P. Robe& R. Emilioxxi, and E.-E. Baulieu 23

were counted (see “Results”). The 14C :3H values were always determined on purified samples; on several occasions the 14C:3H ratio was >I, and it is evident that in these cases the value is only approximate due to the characteristics of double isotope counting.

The R value, defined as the ratio of the r4C:3H (or 3H:14C) ratio of a metabolite divided by the r4C:3H (or 3H:14C) ratio of the injected materials, was calculated when possible. It shows the respective contribution of testosterone glucuronide-14C (-3H) and testosterone-3H (-1°C) to the metabolite under consideration. This is evident since R equals conversion from testosterone glucuronide divided by the conversion from testosterone (2).

Production rates were calculated from the specific activity of urinary testosterone glucuronide by the conventional isotope dilution formula (24).

RESULTS

Structure of Urinary Testosterone Glucuronide

Extraction-Hydrolysis Experiments

These were performed with testosterone glucuronide, radioac- tive testosterone glucuronide, and 5-androstene-3/3,17P-diol diglucuronide obtained from 5-androstene-3/3,17&diol diglu- curonide triacetate methyl ester (922.95) ; m.p. 271-272”; [(y]E30 = -53 f 4” (c, 1% in dioxane).

C45020&

Calculated: C 58.57, H 6.77 Found : C 58.63, H 6.84

This diglucuronide was used as a model compound because testosterone diglucuronide (2,4- (or 3,5)-androstadiene-3, 17&diol diglucuronide) was not available. Testosterone 3-enol- glucuronide was not available either, and androst-5-ene-3P, 17& diol 3-glucuronide obtained by hydrogenation of 3/%hydroxy- androst-5.ene-17-one glucuronide (12) was used as a model compound for testosterone 3-enol-glucuronide.

Testosterone glucuronide was completely ext.racted with ethyl acetate from urine or water solutions containing up to 0.25 mmole per liter. The solutions were acidified to pH 1 with sulfuric acid, NaCl was added to 20% (w/v), and rotating extraction was carried out (3 times for 1 hour) with an equal volume of ethyl acetate. Under the same conditions, approxi- mately half of the diglucuronide was extracted. The organic and the aqueous phases were studied by paper chromatography after /3-glucuronidase (Ketodase or Helix porn&z enzyme) hy- drolysis. The extraction with n-butyl alcohol from water or urine (pH 7) was total for testosterone glucuronide and nil for the diglucuronide.

Urinary Testosterone Glucuronide Isolation-Urine was col- lected for 4 days after the injection of testosterone-7ar-3H (33 PC, Radiochemical Centre Amersham, TRA 165, Batch 1, specific activity 2.9 mC per mg) into a man, and it was extracted at pH 7 with n-butyl alcohol.

The n-butyl alcohol extract was chromatographed in System I. The glucuronides eluted with acetone-methanol (50 : 50) and (25: 75), and with methanol were chromatographed with System J. Then testosterone-3H-14C glucuronide was separated from other 3H-labeled glucuronides in Systems K and L. Urinary testosterone-3H glucuronide, isolated with the use of a testos- terone-14C glucuronide tracer, was hydrolyzed, and the labeled

testosterone, isolated by gradient partition chromatography, was found to contain 0.7% of the injected activity.

All of the eluates of the Celite chromatography of the glu- curonides were pooled with the exception of the testosterone glucuronide fraction just studied. Aft.er fi-glucuronidase hy- drolysis, the free steroids extracted with dichloromethane were chromatographed and a maximum of 0.01% of injected testos- terone-3H was tentatively identified. Since no testosterone 3-enol-glucuronide was available, it was believed that possible endogenous 3-enol glucuronide would have been eluted from the Celite column in approximately the same way as the model 5-androstene-3@, 17@-diol 3-glucuronide. Thus testosterone measured in the present experiment indicates the maximal value of an endogenous production of testosterone enolglucuronide from testosterone.

The urine, after n-butyl alcohol extraction, was hydrolyzed with /3-glucuronidase and no testosterone was found by a subse- quent dichloromethane extraction. Thus, it may be concluded from this part of the studies that no testosterone diglucuronide is excreted into the urine in normal man.

A subsequent 15-min hydrolysis of the urine, at 100” with 10% HCl, was performed and was followed by dichloromethane ex- traction, gradient partition (System E), and paper (System F) chromatography. Some radioactive testosterone, representing 0.06% of the injected activity and less than 0.1% of urinary radioactive testosterone 17-glucuronide was tentatively identi- fied. This acid-hydrolysable testosterone does not invalidate the specific activity determination based on the isolation of urinary testosterone freed by fl-glucuronidase.

Comparison between Enclogenous and Injected Testosterone Glu- curonide-An experiment was performed on the urinary metab- olites of Experiment 3. The urine was extracted with ethyl acetate according to the procedure reported in Fig. 1; part of the alkaline washings (“glucuronide fractions”) was extracted with n-butyl alcohol; the rest hydrolyzed as usual (18). Radioactive testosterone was isolated from the butyl alcohol extract by gradient partition Systems K and L. Since in this experiment testosterone-l ,2-3H glucuronide and testosterone-4-14C have been injected, any testosterone-3H-14C glucuronide isolated as a con- jugate from the butyl alcohol extract should have had the same 3H:14C ratio as testosterone-3H-14C freed by /3-glucuronidase, if testosterone 17-glucuronide was the only test.osterone glu- curonide formed from testosterone-14C and hydrolyzed by p- glucuronidase. If another glucuronide of testosterone-14C were present, it would have contained more i4C in the testosterone liberated with ,&glucuronidase than in testosterone isolated as testosterone 17-glucuronide. Results showing the same 3H:W ratio (3.01 and 3.09) in both isolated testosterones confirmed that no glucuronide of testosterone other than 17-glucuronide has any quantitative importance and therefore this validates the studies herein presented.

METABOLIC EXPERIMENTS

Urinary Metabolites in Three Men and One Pregnant Woman

The results of Experiments 1 to 4 are given in Tables II, III, IV, and VIII.

Isolation of Urinary @-Steroids

These were labeled by both C-14 and tritium. S/S-Androsterone in Glucuronide Fraction-The ketonic fraction

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24 Studies on Testosterone Metabolism. III Vol. 241, No. 1

TABLE II

Intravenous kiection o.f testosterone-3H and testosterone-‘% glucurkde to-normal man (Experiment 1)

Urinary metaboliteP

Androsterone (sulfate). E;piandrosterone (sulfate). Androsterone (glucuro-

nide).................... Androstane-3cy, l’lp-diol

(glucuronide)

5p-Androsterone (sulfate). 5&Androsterone (glucuro-

nide)................... 5p-Androstane-3a, 17p-diol

(glucuronide)

Testosterone (glucuronide).

-

C fi

-

onversion’ mm testes terone-3H

onversiod ram testes :WXX-‘4C lucuronide

% %

4.2 <O.l 1.2 <O.l

4.1 <O.l

1.2 <O.l

0.7 0.4

5.2 6.0

2.7 8.7

N.D.d 16.2

‘C: 3H

<O.Ol <O.Ol

<O.Ol

-0.05

0.8

1.7

4.5

N.D.

-

-

R=

<O.Ol <O.Ol

<O.Ol

<O.l

0.G

1.2

3.3

N.D.

a Actually steroids freed from their sulfate or glucuronide, respectively (see “Urine Analyses”).

fi Conversion of the injected compound to the urinary metabo- lite.

c See “Counting and Calculation.” d N.D., not determined.

TABLE III

Intravenous injection of testosteroneJH and testosterone-W glucuronide to normal man (Experiment 2)

Production rates: testosterone, 8.6 mg; testosterone glucuro- nide, 0.38 mg.

Urinary metabolitesa

Androsterone (sulfate). Epiandrosterone (sulfate). Androsterone (glucuro-

nide)................... Androstane-3a, 17p-diol

(glucuronide)

5@-Androsterone (sulfate). 5p-Androsterone (glucuro-

nide)................... 5fi-Androstane-3a, 17p-diol

(glucuronide)

Testosterone (glucuronide)

?I --

onversior :onl test0 terone-3H

% 3.6 1.4

7.1

0.9

0.7

5.3

1.8

0.6

hversionz mm testes terone-‘4C :lucuronid~

% N.D.d N.D.

N.D.

N.D.

N.D.

3.8

4.3

11.0

N.D. N.D.

N.D.

N.D.

N.D.

0.05

0.16

1.28

RC

<O.Ol <O.Ol

<O.Ol

<O.Ol

N.D.

0.7

2.3

18.3

a Actually steroids freed from their sulfate or glucuronide, respectively (see “Urine Analyses”).

b Conversion of the injected compound to the urinary metabo- lite.

c See “Counting and Calculation.” d N.D., not determined.

of the glucuronide extract was chromatographed in System E. The tubes of the 5/Landrosterone peak were collected, and after evaporation of the solvents, the residue was chromatographed in System A. After pooling the peak tubes, some authentic 5@-

androsterone was added to an aliquot and, after crystallization from methanol-water, crystals and mother liquors were analyzed, confirming the radiochemical homogeneity of the supposed 5&androsterone-%-3H (Table V). Further confirmation of this identity was obtained from thin layer (System C) and paper (System F) chromatography. A special effort was made during these four different chromatographic studies to separate 5@- androsteroneKYH from the other steroids with an equatorial hydroxyl at the C-3 position (epiandrosterone), and from 17p- hydroxy-5fi-androstan-3-one, a compound conceivably biosyn- thesized during the metabolism of testosterone glucuronide and which has a polarity very close to that of 5@androsterone. Neither labeled epiandrosterone nor radioactive 17&hydroxy- 5&androstan&one was detected.

Moreover, the possibility of the occurrence of radioactive 3a- or 3.6hydroxy-androst-4-ene-17-one was checked, these com- pounds being theoretically possible metabolites of testosterone and testosterone glucuronide with polarities closely similar to that of 5B-androsterone. To part of the dried mother liquors of the crystallization reported in Table V, 0.1 mg of androst-4-ene- 3/3,17&diol was added. The mixture was dissolved in 5 ml of CHC13 to which 10 mg of MnOz were then added, and the suspen- sion was shaken overnight (25). After filtration and evapora- tion of the solvent, the residue was dissolved in methanol and the yield of the oxidation product was determined from the absorption of testosterone formed from the added androst-4-ene- 3@, 17@-diol at 240 rnp. Since, in the same manner, any radio- active 3~ or 3/3-hydroxy-androst-4-ene-17-one would have given labeled androst-4-ene-3,17-dione, the extract was chromato- graphed on paper (System F) and the four regions of androst-4- ene-3,B ,17p-diol, 5/Landrosterone, testosterone, and androst-4-

TABLE IV

Intravenous injection of testosterone-%’ and testosteroneJH glucuronide to normal man (Experiment 8)

Production rates: testosterone, 6.9 mg; testosterone glucuro- nide, 0.4 mg. -

Urinary metabolitesa onversiont ‘OnI testes .SXle-‘4C

,C -f

i __

8H:W RC

Androsterone (sulfate). Epiandrosterone (sulfate). Androsterone (glucuro-

nide)................... Androstane-3ol, 17p-diol

(glucuronide) .

5p-Androsterone (sulfate). 5&Androsterone (glucuro-

nide)................... 5p-Androstane-3a, 17p-diol

(glucuronide) . .

N.D. N.D. N.D. N.D.

N.D. N.D.

N.D. N.D.

0.1 0.4

0.2 1.0

1.1 5.6

2.9 15.6

a Actually steroids freed from their sulfate or glucuronide, respectively (see “Urine Analyses”).

6 Conversion of the injected compound to the urinary metabo- lite.

c See “Counting and Calculation.” d N.D., not determined.

Testosterone (glucuronide)

C’ h t

-

%

7.6 2.6

14.1

1.8

2.7

9.3

2.9

1.5

:onversion” mm testes terone-aH :lucuronid~

%

N.D.4 N.D.

N.D.

N.D.

1.2

9.6

16.3

23.5 -

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Issue of January 10, 1966 P. Robe& R. Emiliozxi, and E.-E. Baulieu 25

ene-3,17-dione were analyzed. In the androst-4-ene-3,17-dione region, little if any tritium was found and the lack of 14C indi- cated the absence of 3a- or 3P-hydroxy-androst-4-ene-17-one-W in the fractions identified as 5&androsterone-14C. The 5& androsterone 14C :3H ratio was unchanged by the MnOz oxidation.

The acetyl derivative of 5/3-androsterone-14C-3H was made. It migrated as authentic 5/%androsterone acetate on paper.

Hydrogenation of another portion of 5fi-androsterone-W-3H with potassium borohydride gave a 14C-3H labeled compound with the same migration pattern as authentic 5,&androstan-3a, 17@diol in System C. The ratio of 14C:3H remained essentially constant in the 5/3-androsterone-W-3H derivatives.

S&Androsterone in Sulfate Fraction-The ketonic fraction of the solvolyzed extract was chromatographed with a Celite parti- tion gradient (System E). The contents of the tubes containing 5B-androsterone and epiandrosterone were pooled and chromato- graphed on an alumina column. A portion of the supposed 5,@ androsterone-14C-3H was mixed with authentic 5&androsterone and crystallized from benzene-isooctane. Proof of its radio- chemical homogeneity was obtained (Table V). Oxidation of some crystals obtained in this last step with MnOz yielded the same results as those obtained in the corresponding 5B-andros- terone glucuronide. The acetate had the same chromatographic behavior as authentic 5&androsterone acetate and the 14C:3H ratio was the same as that of the free compound.

5&Androstan-.%, 17p-dial in Glucuronide Fraction-The non- ketonic fraction of the glucuronide extract was chromatographed on alumina according to System B. The peak fractions were combined and a small aliquot was chromatographed on paper with System G. The only radioactive spot behaved like au- thentic 5P-androstan-3a!, 17/?-diol. The remainder was chro- matographed on a Celite colume (10 g) in the isooctane-ethyl acetate-methanol-water (8:5:9: 1) system and almost all of the radioactivity was found in a single peak. Authentic 5/?-a,- drostan-3a!, 17&diol was added to part of the contents of the peak tubes and successive crystallizations were performed (Table VI).

Another fraction of the radioactive peak of the partition chro- matography was submitted to MnOz oxidation after addition of 0.1 mg of androst-4-ene-3/3,17@-dial. Testosterone formatlion was detected by ultraviolet absorption, but after its isolation by paper chromatography (System G), it was found not to be radioactive, indicating that no radioactive androst-4-ene-3a, 17@-diol or androst-4-ene-3P, 17@-diol were present in the supposed 5fi-androstan-Qa , 17P-diol-14C-3H. MnOp oxidation left the 1% :3H ratio of 5&androstane-3a, 17&diol unchanged as

TABLE V

Identification of radioactive 5&androsterone in Experiment 1

W 3H WZ: 8H .,

5p-Androsterone (glucuronide)a Theoretical. Crystals. Mother liquors..

5.B.Androsterone (sulfate)a Theoretical. Crystals. Mother liquors..

cm/w

2000 1120 2060 1170 1960 1040

545 720 500 660 500 690’

1.8 1.8 1.9

0.8 0.8 0.8

a Actually steroids freed from their sulfate or glucuronide, respectively (see “Urine Analyses”).

TABLE VI

IdentQication of radioactive 5P-androstan-Scr,lrp-diol (ylucuronide) in Experiment 1

Added 5p-androstane-3cu, 17fl-diol w-eight, 10.00 mg; theoretical specific activity, 1100 cpm per mg.

1st crystallization (methanol-water) Crystals. Mother liquors......................

2nd cryst,allization (benzene-isooctane) Crystals. Mother liquors..

3rd crystallization (methanol-water)* Crystals. Mother liquors..

Keight of Specific activity of

sample C-14a Tritium

2.19 990 0.92 930

0.51 1090 0.72 1020

0.37 980 0.30 1010

w @dW

/ -

220 230

Q Due to the relative insolubility of 5p-androstane-3a, 17@diol in the scintillation solution, 2 ml of methanol were added for counting the crystals and mother liquors in the 1st and 2nd crystallizations, and 0.2 ml was added in the 3rd crystallization. In all of the cases, the obtained value for both tritium and C-14 was corrected for quenching. The calculations seemed valid for tritium only in the 3rd crystallization, when counting was per- formed with little methanol.

* In the 3rd crystallization, the 14C:3H ratios were 4.6 for crys- tals and 4.3 for mother liquors.

far as it was possible to judge by counting the steroid eluted from the paper chromatogram. In addition, another fraction of the peak from the partition column was acetylated and chromato- graphed on paper in the ligroin-propanediol and heptane-phenyl- cellosolve solvent (System F); the radioactive product migrated like authentic S/3-androstan-Qa! ,17&diol diacetate.

Isolation of Urinary Sa-Metabolites

The urinary 5cu-metabolites contained only the same label as the injected testosterone.

Androsterone in Glucuronide Fraction-The first radioactive peak of the gradient partition chromatography of the ketonic fraction (System E) was studied. After evaporation of the solvents, the material was chromatographed on alumina with System A. The only peak had the polarity of androsterone. Results of crystallizations from methanol-water after addition of authentic androsterone confirmed both the identity of the an- drosterone and its very low 14C :3H ratio in Experiments 1 and 2, and 3H:14C in Experiment 3.

Androsterone in Sulfate Fraction-The androsterone peak after chromatography of the ketonic extract in System E was purified further by alumina gradient chromatography. Its identity was carefully checked by paper chromatography (System F), and the absence of radioactive 17&hydroxy-5cY-androstan-3-one was ob- served. Another part of the peak was mixed with pure andros- terone and crystallized from benzene-isooctane; the identical specific activities of the crystals and the mother liquors confirmed the homogeneity of the isolated product.

Epiandrosterone in Sulfate Fraction (Tentative Identi$cation)- The second peak of the partition gradient chromatography of the ketonic sulfate fraction (System E) was further purified by

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26 Studies on Testosterone Metabolism. III Vol. 241, No. 1

TABLE VII

Identijkation of radioactive testosterone (glucuronide)

in Experiments 1 and & (1-P)

Testosterone (glucuronide) ‘4C 3Ha ‘C: aH

wdw Experiment 1

Theoretical. Crystals. Mother liquors..

Experiment 2

240 215 225

Theoretical. 275 285 1.0 Crystals. 285 295 1.0 Mother liquors.. 255 270 0.9

(1 In Experiment 1, 3H was not visible in testosterone glucuro- nide; therefore, the injected 3H:14C was changed in Experiment 2.

TABLE 1'111

Intravenous injection of testosteroneJYT glucuronicle to a pregnant woman (Experiment 4) and intramuscular injection of testosterone-14C and testosterone glucuronide to a child

(Experiment 5)

Injected compounds

Experiment 4 Testosterone-%

glucuronide. Experiment 5

Testosterone-14C. Testosterone glu-

curonide. .

-

1

.-

-

Indrosterone

-

-

56-Andro- 56-Andr

sterone tan-3n, 1 diol

os- 7P- Testosterone

% % % %

<O.l 3.8 8.2 2.9

5.9 3.0 1.0

so.3 2.8 20.0

Urinary metaboliteP Urinary Metabolites in a Child (Experiment 5)

The determinations of the urinary metabolites in the child in- jected with testosterone-14C and testosterone glucuronide are listed in Table VIII.

a Results are espressed as counts per min in the metabolite/ counts per min injected X 100.

alumina chromatography (System A). The identity was con-

firmed by paper chromatography (System F). Androstane-S’ol , I?+@-dial in Glucuronide Fraction-The non-

ketonic fraction of the glucuronic extract was chromatographed on Column B. An aliquot migrated on paper in System G at the same speed as authentic androstane-3a, 17@-diol. The rest was purified further on Celite colume (10 g) in the ethyl acetate- isooctane-methanol-water (4: 8 : 9: 1) system. Part of it was mixed with authentic androstane-3a ,17fl-diol and crystallized from methanol-benzene; the specific activity of the crystals and the mother liquors was the same.

Testosterone in Glucuronide Fraction

The ketonic fraction of the glucuronide extract was chromato- graphed in the gradient partition (System E). The testosterone peak was purified with gradient alumina (System H), and the specific activity was checked in peak tubes (Experiment 2, specific activity (counts per min per Hg): 3H, 111, 126, 117, 116; 14C, 148, 157, 156, 146). Crystallization from isooctane-benzene con- firmed the radiochemical purity of the isolated compound (Ta- ble VII).

Calculations

In Experiment 1, the 3H:W ratio in urinary testosterone glu- curonide did not allow calculation of the conversion of injected testosterone to urinary testosterone glucuronide. Moreover the injection of more than 2 mg of testosterone-l*C in the form of glucuronide made determination of the endogenous production of testosterone glucuronide unreliable. Experiments 2 and 3 were devised in order to determine the contribution of both injected radioactive testosterone and t,estosterone glucuronide to urinary labeled testosterone. The weights of the injected labeled testos- terone glucuronides were low enough (0.2 and 0.09 mg, respec- tively) to allow calculation of the production rates from the specific activity of urinary testosterone glucuronide, the quantity of which was measured by ultraviolet absorption at 240 rnM in methanol. The weight of the injected compounds has been taken into account in the calculations.

In Tables II, III, and IV the isotope ratios vary greatly in each experiment from one metabolite to another, and consequently they have not the same accuracy (e.g. the ‘4C:sH ratios <O.l must be taken only as approximate). The injected ratio was changed in each experiment in order to overcome this difficulty arising from the nature of the metabolic steps being studied.

Paper chromatography of a part of the glucuronide fraction revealed testosterone as a radioactive ultraviolet-absorbing zone. It was further identified by pooling the tubes of the appropriate zone of the gradient chromatography on alumina of the rest of

the glucuronide extract and rechromatography of the residue on a Celite column (25 g) in the isooctane-ethyl acetate-methanol- water (22:8:25:5) system. The correlation between the ultra- violet and the radioactive curves was very good. The percentage of excretion of testosterone glucuronide into the urine is of the same magnitude as that obtained in the adult experiments. It may be added that testosterone-14C is converted to urinary testosterone-14C glucuronide in the same proportion as it is in adults.

5B-Androsterone was detected after paper chromatography of a fraction of the glucuronide extract of urine obtained 2 days after the injection. It was isolated after gradient alumina chroma- tography of the rest of the glucuronide extract, the peak being detected by the 14C curve, and the pooled material was chromato- graphed on paper in the System F. The eluate of the 5/3-an- drosterone zone was purified through a Celite-silica gel (1:l) column (2 g) from which it was eluted with benzene-ether mix- tures. After drying, the residue was analyzed by infrared spec- trophotometry which confirmed the identity of 5fi-androsterone. The nonketonic compounds were not studied.

The conversion rate of testosterone glucuronide to urinary 5fi-androsterone in the child is less than the corresponding trans- formation in adult experiments. The conversion of the testos- teroneJ4C tracer to radioactive androsterone and 5fi-andros- terone glucuronides shows a preponderance of the 5a form.

DISCUSSION

This work provides evidence that testosterone glucuronide is further metabolized in the body. Moreover this metabolism,

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Issue of January 10, 1966 P. Robe& R. Emilioxxi, and E.-E. Baulieu 27

unlike that of testosterone, is specifically oriented towards the production of 5p-metabolites (Fig. 2). The production of testos- terone glucuronide, and therefore the formation of testosterone glucuronide from testosterone, is a relatively minor phenomenon.

Such results depend on some technical assumptions and lead to further conclusions concerning testosterone glucuronide metab- olism, qualitatively and quantitatively.

Technical Assumptions

The validity of these studies depends on the assumption that the injected tracer is identical with the endogenous compound.

Injected testosterone glucuronide is assumed to have the /?- glucopyranuronoside structure with linkage at the C-17 position, since the mode of synthesis that was used has been known for a long time to give this structure (13). Ultraviolet absorption at 240 rnp of the A4-3-ketone group and complete hydrolysis with P-glucuronidase served as controls; the latter confirmed the ,&glucuronide structure and demonstrated that the sugar ring was pyranose since @glucofuranuronosides are not hydrolyzed by the enzyme (21).

The structure of endogenous testosterone glucuronide in man is not well established. Blood testosterone glucuronide (11) and urinary testosterone glucuronide (4) are defined, in regard to t,heir structure, by hydrolysis with P-glucuronidase. Thus, there is no distinction between the conventional ,&glucuronide at the C-17 position, the possible /3-glucuronide of an enol at C-3, and even a diglucuronide (C-17 + C-3 enol). Edwards and Kellie (26), after administration of large doses of testosterone to a nor- mal man, obtained urinary testosterone glucuronide from the ketonic fraction of a “conjugate extract,” acetylated and methylated, with the use of the Girard T reagent; therefore, the glucuronide of the enol form of testosterone and the diglucuronide are excluded.

In the present experiments, the specific activity of urinary testosterone was measured after the injection of radioactive testosterone 17-glucuronide; it would be decreased by the pres- ence of any glucuronide of the enol form, should the enzymic hydrolysis cleave the enol as well as the C-17 form. Thus, the calculated production of testosterone glucuronide would be ar- bitrarily elevated; the same problem could be raised if there was some testosterone diglucuronide not derived from testosterone 17-glucuronide. The present studies show that if the enol form or the diglucuronide exist at all, they do not have any quantita- tive importance in normal people.

Therefore, from the absence of any other form of testosterone glucuronide in urine, it is believed that testosterone glucuronide circulating in the blood (10) has the C-17 structure. Thus, the metabolic studies have a physiological significance and it is possi- ble to calculate the production rate of testosterone glucuronide from the specific activity of urinary testosterone obtained by /3-glucuronidase hydrolysis, after the injection of radioactive testosterone 17-glucuronide.5

The amount of testosterone glucuronide injected in Experi- ments 1, 2, and 5 (Table I) was relatively large, considering the calculated production of testosterone glucuronide of 0.5 mg at the most in normal men. However, Experiment 3 with a tracer

5 Note Added in Proof-Hadd and Rhamy (J. Clin. Endocrinol. and Metabolism, 25, 876 (1965)) have given evidence for the 17p + lp-D-glucuronide structure of human blood testosterone glucuro- nide, in agreement with the present urinary studies and validation of the metabolic significance of this work.

dose indicates that the 5&metabolism of testosterone glucuronide is not a pharmacological phenomenon. It is also very significant that in the child (Experiment 5, Table VIII), a large dose of testosterone glucuronide gives 5&androsterone and no andros- terone at all, whereas the simultaneous injection of testosterone- 14C indicates that the testosterone metabolism is directed mainly towards the formation of the 5B-compounds, as is usual in chil- dren.

Metabolism of Testosterone Glucuronide

Qualitative Considerations

I f the steroid conjugates, considered classically as end products, are not totally excreted as such and yield one or more metabolites, this may be termed “further metabolism” (27). With sulfates, these metabolites can be formed “directly” without cleavage from the acid moiety. This is rigorously established by the trans- formation in viva of steroid sulfate labeled with tritium and 3% into another doubly labeled steroid sulfate (28, 29) ; the metabo- lites can be also formed “indirectly” after a split of the conjugates (see References 30 to 32).

Concerning the glucuronides, it is evident that different glu- curonides do not have an identical metabolic fate. Androsterone glucuronide does not undergo any metabolism (9). Dehydro- epiandrosterone glucuronide gives androsterone and 5&andros- terone glucuronides but, after oral administration (33), an attack by digestive enzymes or bacteria could have occurred and the physiological significance of the results is uncertain. Recently (32), the urinary metabolites of radioactive epiandrosterone glu- curonide and radioactive dehydroepiandrosterone glucuronide injected intravenously to normal people were analyzed and a further metabolism of these C-3 /?-hydroxy-glucuronides, includ- ing a cleavage of the glycosidic link, was shown.

The absence of 5ol-metabolites from testosterone glucuronide in the present experiments (Fig. 2) leads one to postulate a lack of hydrolysis of testosterone glucuronide to testosterone and a sub- sequent direct further metabolism of testosterone glucuronide. An unlikely alternative is that testosterone is freed from testos- terone glucuronide in compartment or compartments from which it could not pass into the blood stream and in which it gives only 5fi-metabolites. However, sooner or later, a cleavage of the glucuronide link must occur in order to give 5/3-androsterone, which is ketonic at the C-17 position and thus must be formed after a break of the C-17 glycosidic link. Therefore, it may be suggested that testost.erone glucuronide does not give testosterone but gives glucuronide or glucuronides, later split to give 5p- androsterone.

The percentage of conversion of radioactive testosterone glu- curonide to urinary labeled 5&androstane-3ar, 17@-diol is greater than that to urinary radioactive 5&androsterone, whereas in the same subjects the conversion of the testosterone tracer to S/3- androstan-3oc,l7/%diol is less than half of the conversion to 5/3- androsterone. On the other hand, the conversion of 5/3-a,- drostan-3a!, 17&diol to 5/%androsterone is probable (2). Thus, it is conceivable that 5&androstan3or, 17&diol arises from testosterone glucuronide and is the precursor of 5/?-androsterone. The conjugation with glucuronic acid of 5&androstan-3oc, 17/L diol is not well defined; it could be a conjugation at either the C-3 level, the C-17 level, or both (diglucuronide). Even a combined sulfate and glucuronide, (e.g. C-17 glucuronide, C-3 sulfate) is not formally excluded, and in this connection it should be pointed

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28 Studies on Testosterone Metabolism. III Vol. 241, No. 1

A H

FIG. 2. Selective 5p-metabolism of testosterone glucuronide. T, t’estosterone; G, glucuronide; A, androsterone; @-A, 5p- androsterone; Adiol, androst’an-~oc,17p-diol; Sp-Adiol, 5fi-androstan-3cu,l7~-diol; TG, testosterone glucuronide.

out that the R of 5&androsterone sulfate is consistently one-half that of 5/Sandrosterone glucuronide. This is evidence that two precursors can contribute differently to the two conjugated forms of a single metabolite. It is entirely possible that 5fi-androstane- 3cr,17pp-diol glucuronide at the C-17 position is a direct me- tabolite of testosterone glucuronide and that 5,Bandrosterone is derived from it but other metabolic pathways are conceivable; the present results suggest that any intermediate cannot be trans- formed into 5or-metabolites and cannot be aromatized in the placenta (Experiment 4).

Further studies are required to determine if the 5/S-metabolism of testosterone glucuronide is due to the very nature of testos- terone glucuronide as a substrate of A4-reductase or A4-reductases, or to the fact that the testosterone glucuronide enters a special “compartment” where testosterone is freed and gives only 5/L steroids. Ss a third possibility, the 5jSmetabolism of testos- terone glucuronide could be due to the intracellular location of the 5~ and 5@-reductases (34), the latter only being reached by the water-soluble glucuronide.

Quantitative Considerations

SigniJicance of Determination of Testosterone Glucuronide and Testosterone Production Rate-If one assumes that intravenously injected testosterone glucuronide traces testosterone glucuronide formed in the liver from any precursor, it is possible to measure validly testosterone glucuronide produrtion rate. This con- sideration may eventually apply to the kidneys in which glucuro- conjugation occurs (35), but in the latter case the calculation could be complicated by a possible total or partial direct excretion of testosterone glucuronide into the urine. If we compare the approximately 0.5 mg per day production rate of testosterone glucuronide found in these studies and the 7 to 9 mg production rate of testosterone found in young normal men by the “blood method” (36), it seems likely that this production rate of testos- terone glucuronide is accounted for entirely by the 5% conversion

TC "secretion"

------Ad T d TG 1

’ Y,

x1

FIG. 3. Respective contribution of t,estosterone and testos- terone glucuronide to a urinary metabolite. T, testosterone; TG, testosterone glucuronide.

of t’estosterone to testosterone glucuronide calculated from the R value of urinary testosterone glucuronide after simultaneous in- travenous injection of radioactive Oestosterone and testosterone glucuronide. This does not favor a secretion, even minimal, of testosterone glucuronide in man, which would increase dra- matically the measurement of testosterone production rate by the urinary method. Kowever, testosterone glucuronide has been found in salmon testes (37).

The production rate of testosterone cannot be established on t’he basis of the specific activity of urinary testosterone glucuro- nide after injection of radioactive testosterone since any small secretion of testosterone glucuronide (of the order of 0.1 mg) would greatly influence the result,, especially in women.

Calculation of Respective Contribution of Testosterone and Testosterone Glucuronide to 5@-AFetabolites--In Fig. 3, u = over-all conversion of testost’erone to a certain urinary metabolite; r/1 = conversion of testosterone to this urinary metabolite via pathways other than testosterone glucuronide; y2 = conversion of

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Issue of January lo,1966 P. Rebel, R. Emilioioxzi, and E.-E. Baulieu 29

testosterone to this urinary metabolite via testosterone glucuro- nide; ~1 = conversion of testosterone glucuronide to the urinary metabolite; Q = metabolic conversion of testosterone to testos- terone glucuronide, thus

y = y1 + y2; Y? = Xl x x2

If we apply these figures to testosterone glucuronide formed from testosterone

y1 = 0; y = yZ = z1 X sZ; 22 = X = 0.05 (experimental) Xl

This value of x2 is used to calculate the quantity of the metab- olite of testosterone formed via testosterone glucuronide.

&&Androsterone-Both testosterone and testosterone glucuro- nide give 5&androsterone with approximately the same yield

CR - 1) ; therefore, x1 = y. The fraction of 5/?-androsterone formed from testosterone via

testosterone glucuronide is

Xl x x2 2 = - = x2 = 0.05 Y Y

This means that only 5% of the 5/I-androsterone formed from testosterone is biosynthesized via testosterone glucuronide.

It can be seen that an R value of 1 (i.e. the same conversion rate of two precursors to a certain metabolite, determinable by the same 3H:14C ratio of the injected precursors of the metabolite) does not prove a metabolic sequence. This was previously ob- served (1,2) in connection with the testosterone to 4-androstene- 3,17-dione to 17-ketosteroid sequence. The demonstration of a 17&hydroxyl pathway of testosterone led to a revision of the previous postulation of a practically 100% conversion of testos- terone to androst-4-ene-3,17-dione.

S/3-Androstan-Sor ,17&dioZ-Similar calculations indicate that approximately 25% of urinary 5/3-androstane-3a, 17&diol formed from testosterone derives from testosterone glucuronide.

Most, if not all, urinary 501- or @-androstane-3a, 17&diols metabolites of testosterone are formed without passing through a 17-ketosteroid intermediate (“17~-hydroxyl pathway”) (1). The present experiments suggest that part of testosterone can possibly be converted to 5/3-androstan-aar , 17/3-diols via testos- terone glucuronide. This “d&our” would keep intact the 17/3- hydroxyl group and could contribute to make the 5@:5a ratio of the androstan-3a!, 17j3-diols greater than that of the 17-ketos- teroids.

Finally, it must be stressed once again that the physiological significance of all of the conversion rates, namely XI and y, and indirectly z2, depends on the mixing of the injected tracers with the natural compounds; if a total mixing does not occur, one must write y’ = uy (instead of y) and ~‘1 = bx1 (instead of XI), a and b being coefficients of entry of the injected compounds into the “metabolic” pool.

Acknowledgments-We acknowledge the valuable technical help of Pierre Rocher. The manuscript was prepared by Anne Bacquet. We thank Misses Maxine Groffsky and Susan Hof- mann for their help in writing the manuscript.

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Paul Robel, Roméo Emiliozzi and Etienne-Emile Baulieu-METABOLISM" OF TESTOSTERONE GLUCURONIDE

βStudies on Testosterone Metabolism: III. THE SELECTIVE "5

1966, 241:20-29.J. Biol. Chem. 

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