STEROID METABOLISM IN GUINEA PIGS

I. METABOLISM OF CORTISOL IN VIVO*

BY SHLOMO BURSTEIN AND RALPH I. DORFMAN

WITHTHETECHNICALASSISTANCEOFARLINE M. TILLOTSONANDPAUL R. SKOGSTROM

(From the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts)

(Received for publication, August 25, 1954)

In previous studies (l-3) cortisol (hydrocortisone) was isolated and identified from the urine of the normal and the scorbutic guinea pig. How- ever, paper partition chromatography of extracts of guinea pig urine (2) revealed that in addition to cortisol several other blue tetrazolium-reduc- ing steroids were present. The possibility existed that some of these steroids were metabolites of endogenous cortisol, and it was believed that an understanding of the metabolic pathways operating in this animal would aid in the evaluation of corticosteroid excretion patterns under nor- mal and pathological conditions. The study of the metabolism of cortisol in this animal therefore was undertaken.

Methods

Steroid Administration and Urine Extraction-Cortisol’ was given orally to normal adult male guinea pigs. Urine was collected for 17 hours fol- lowing steroid feeding, during which time more than 90 per cent of the metabolites containing the ar-ketol side chain were excreted. The urine was extracted with ethyl acetate after adjustment to pH 6 by the addition of acetic acid to dissolve the precipitate present in guinea pig urine.

Paper Partition Chromatography-The crude extracts were fractionated by paper chromatography, as previously described, with the chloroform- formamide and the toluene-propylene glycol systems (4, 5). The indi- vidual zones were detected by the blue tetrazolium reagent and by ex- amination of the chromatograms under ultraviolet light (Mineralight, Ultra-Violet Products, Inc., South Pasadena, California). The quanti- tative paper chromatographic procedure with ultraviolet spectrophotom- etry described previously (2) was employed for the quantitative determi-

* This work was supported in part by a research grant, NSF-G664, from the Na- tional Science Foundation, and by contract No. AT(30-l)-918 with the United States Atomic Energy Commission. A preliminary abstract has been presented at the 126th meeting of the American Chemical Society, New York, September 14, 1954.

1 Thanks are due to Dr. K. Pfister for the gifts of cortisol, 6fi,21-diacetoxy-17a- hydroxy-4-pregnene-3,11,20-trione, and 11@,17a,U)or,21-tetrahydroxy-4-pregnen-3- one.

581

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582 STEROID METABOLISM. I

nation of the urinary metabolites after administration of 20, 40, and 8Olmg. of cortisol per animal.

Column Partition Chromatography-Column partition chromatography with acid-washed (6) Hyflo Super-Cel (Johns-Manville, New York) as the supporting phase was used (6, 7). A water-95 per cent ethanol solu- tion 3: 1 (volume per volume) was used as the stationary phase. The mobile phases consisted of organic solvents which were saturated Gith an equal volume of the stationary phase. The mobile phases included petro- leum ether, toluene, benzene, benzene-methylene chloride 15:7, methylene chloride-benzene 15:7, methylene chloride containing 5 per cent butanol, methylene chloride-ethyl acetate 1: 1, and ethyl acetate. All the solvents were redistilled, and no blank values as determined by weight were ob- tained. The fractionation procedure was followed by weighing. 40 ml. fractions were collected on an automatic fraction collector. A ratio of adsorbent to stationary phase 3:2 (gm. per ml.) was used.

Steroid Identification and Determination-The isolated steroids were identified by melting point determinations and by infra-red, visible, and ultraviolet spectrophotometry. All melting points were determined on the Kofler micro hot stage apparatus and are uncorrected. The infra-red spectra2 were recorded on the Perkin-Elmer 12C model and were carried out on solid films deposited on sodium chloride, unless otherwise stated. The absorption coefficients in the ultraviolet were determined on the Beckman DU spectrophotometer and the spectra in sulfuric acid on the Cary recording 11MS spectrometer.

As the spectra in sulfuric acid of the isolated steroids characteristically change with time, they were recorded immediately (within 0.5 minute) and 120 minutes after dissolving the steroid in acid. The two spectra are characteristic of the steroid and adequately represent the change. The average recording time of a spectrum from 220 to 600 rnp was 10 min- utes f 1 minute.

EXPERIMENTAL

Isolation of Cortisol Urinary Metabolites-A total of 2 gm. of cortisol was fed to twenty guinea pigs, 100 mg. per guinea pig. The ethyl acetate extract of the urine was chromatographed in the chloroform-formamide system on twelve 15 cm. wide papers for 17 hours; Fig. 1 illustrates the chromatographic separation achieved. Fig. 2 shows the separation achieved by column partition chromatography of an extract of guinea pig urine following the feeding of 1 gm. of cortisol to ten guinea pigs, 100 mg. per animal. The description of steroid isolation follows.

*We are indebted to Dr. H. Rosenkrantz for advice in the interpretation of the infra-red spectra.

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S. BURSTEIN AND R. I. DORFMAN 583

S/3,1 l/3,1 ~CI, 21 -Tetrahydroxy-4-pregnene-3 ,%‘O-dione (6/3-Hydroxycortisol) @)-This steroid (as the acetate) was isolated from Zone I (Fig. 1) and from Fractions 61 to 66 (Fig. 2). A yellow-brown pigment consistently ran along with Zone I and, despite repeated paper chromatography in the chloroform-formamide system, no separation of the steroid from the pig- ment was achieved. As no purification of the bulk of the material was

ctp : I :II III

10. Iv V VJ

20 .

40 . VIJJ

FIG. 1. Diagrammatic presentation of a paper chromatogram (run in the chloro- form-formamide system for 17 hours) of the ethyl acetate extract of guinea pig urine after the oral administration of cortisol. Zones I to VIII were detected by examina- tion under ultraviolet light. The reducing steroid zones were detected on strips cut along the sides of the paper by the blue tetrazolium color reaction, and the intensities of the observed blue colors are represented by the different shades. The major components of Zones I, IV, V, and VII were identified as 6@, l&3,1701,21-tetrahydroxy- 4-pregnene-3,20-dione, llp,17or,20~~,21-tetrahydroxy-4-pregnen-3-one, 11~,17~u,20~,- 21-tetrahydroxy-4-pregnen%one, and cortisol, respectively. The location of the zones is indicated on the scale in cm. from the starting line (the double line at 0 cm.).

possible by crystallization, the combined crystals and mother liquors com- prising Zone I were acetylated at room temperature in the usual manner. The acetylated mixture was chromatographed in the toluene-propylene glycol system for 17 hours. The yellow-brown pigment stayed at the starting line. A major zone that reduced the blue tetrazolium reagent was observed under ultraviolet light at 9.7 to 14.2 cm. from the starting line. Three other zones in smaller concentrations were detected by ultra- violet scanning, but were not identified. The major blue tetrazolium-re-

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584 STEROID METABOLISM. I

ducing zone was rechromatographed twice on paper and crystallized from acetone, ethyl acetate, and finally from 95 per cent ethanol. Colorless needles were obtained which melted at 122-125’; [a]z3 +82’ (MeOH). Microanalysis indicated an elemental composition of CzsH3408.Hz0, which

&H3609. Calculated, C 62.48, H 7.55; found, C 62.10, H 7.44

is consistJent with the compound, 60,21-diacetoxy-llfl , 17a-dihydroxy-4- pregnene-3,20-dione monohydrate (I-ac.). The molecular extinction co- efficient (X,,. 236 rnp; 6 = 12,300) of the compound is in keeping with

25 PETROLEUM

ETHER 20

TOLUENE

I lo 30 41

LsLl . . . 50 60 70 103 110

FRACTION NUMBER FIG. 2. Chromatographic pattern of a 145 mg. extract from urine collected from

guinea pigs fed 1 gm. of cortisol. Chromatography on Hyflo Super-Cel partition column with water containing 25 per cent ethanol as the stationary phase. Elution with solvents saturated with the stationary phase as indicated. A total of 138 frac- tions of 40 ml. was collected. Fractions 74 to 77 were eluted with CH&X2, Fractions 78 to 86 with methylene chloride containing 5 per cent, butanol, Fractions 87 to 102 with methylene chloride-ethyl acetate 1:l. The elution with ethyl acetate was con- tinued to Fraction 138. No peaks were observed in these fractions.

the molecular extinction coefficients observed for other G@hydroxylated steroids (8). The infra-red spectrum (in a mull with paraffin oil) of this material is given in Fig. 3. The absorption spectrum of the isolated I-ac. in concentrated sulfuric acid is shown in Fig. 4. As can be seen from Fig. 4, the spectrum in sulfuric acid changes with time. However, the two extreme curves, immediately (within 0.5 minute) after (Curve 1) and 120 minutes after (Curve 5) dissolving in acid, represent two characteristic spect,ra of the steroid. Characteristic changes with time were also ob- served in the spectra of the other isolated steroids and are represented by the two extreme curves (see below). On dissolving I-ac. in sulfuric acid, a transient violet-purple color, but no green fluorescence, was obtained.

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S. BURSTEIN AND R. I. DORFMAN 585

CCI,OAC ;H,OAC

oJ$;t;%$.$pu

Identification of the isolated compound (I-ac.) was achieved by mild oxidation to 6/3,21-diacetoxy-17~-hydroxy-4-pregnene-3,11,20-trione (6p- hydroxycortisone diacetate).

WAVELENGTH IN fi 345 6 7 8 9 lo 11 12

I(I,I,I,I(I, 1, 1 j I

z % n w z 4 a I- x

I I I I I I I I 2500 1500 1300 1200 1100 1000 900 850

WAVENUMBER IN CM-’ FIG. 3. Infra-red absorption spectrum of 6@,21-diacetoxy-118,17a-dihydroxy-4-

pregnene-3,20-dione monohydrate in a mull with paraffin oil.

The oxidation with chromic acid was carried out in a manner similar to that described by Reichstein (9) for the oxidation of cortisol acetate to cortisone acetate. To 2 mg. of I-ac. 0.3 ml. of 90 per cent acetic acid con- taining 3 mg. of CrOs was added and the mixture shaken at room tempera- ture for 5 minutes. Following the addition of methanol, the solution was evaporated almost to dryness and extracted with ethyl acetate. The ethyl acetate was washed with water, dried over anhydrous NazS04, and evaporated in VCLCUO. Crystallization from 95 per cent ethanol afforded colorless crystals (rhombohedric rods) which melted at 225-233”. No de- pression in the melting point resulted by the admixture of an authentic sample of G/Shydroxycortisone diacetate (m.p. 228-236”).1 The infra-red spectrum and the absorption spectrum in sulfuric acid (Fig. 5) of the ma- terial were identical with those of the authentic sampIe. In addition, the isolated I-ac. was identical wit,h the acetate of the incubation product

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586 STEROID METABOLISM. I

prepared by Hayano and Dorfman by the incubation of 6/3,17~~,21-tri- hydroxy-4-pregnene-3,20-dione with the 1 Whydroxylase system of beef adrenals (10).

J 1 L 11 11 1 ’ 1 ’ “1 1 #“I”’ 1 &I 260 280 300 320 350 400 450 xx) 550 f

WAVE LENGTH IN MJJ 0

FIG. 4. Changes with time of the absorption spectrum of 6fl,21-diacetoxy-llp,17cu- dihydroxy-4-pregnene-3,2O-dione monohydrate in sulfuric acid (approximately 150 7 in 3 ml.). The spectra are presented as recorded by the Cary 1lMS recording spec- trometer. The average recording time was 10 minutes f 1 minute. Curves 1 to 5 were obtained within 0.5,15,30,60, and 120 minutes, respectively, after dissolving the steroid in sulfuric acid. The curves were plotted one above the other for conven- ience. The optical densities at the maxima 240, 280, 340,390, and 480 rnp were 1.77, 1.83; 1.26, 1.16; 0.54, 1.38; 0.60, 0.80; and 0.45, 0.73 for Curves 1 and 5, respectively. The optical density at the 550 rnp peak in Curve 5 was 0.59.

Hydrolysis of the diacetate monohydrate (I-ac.) to the free steroid (I) with alkali did not proceed smoothly. Herzig and Ehrenstein (8) have shown that 6/Sacetoxy steroids isomerize in aqueous alcoholic alkaline media to the allopregnane3,6-dione derivatives; smooth hydrolysis was shown to proceed in absolute ethanol under nitrogen. By using aqueous

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S. BURSTEIN AND R. I. DORFMAN 587

methanolic KHCOS, I-ac. gave only a poor yield of a material which had the mobility on paper of I. A greater yield of I was obtained when I-ac. was dissolved in oxygen-free ethanol and treated with 2.3 equivalents of ethanolic KOH. Paper chromatography in the chloroform-formamide sys- tem revealed .the presence of four reducing components. The most polar major component was identical in mobility with I and showed a maximal

20

220 240 260 280 300 320 350 400 4% 500 550 600 wave Cet7gtb h rnp

FIG. 5. Absorption spectrum of 6@,21-diacetoxy-17a-hydroxy-4-pregnene-3,11,20- trione in sulfuric acid (approximately 150 y in 3 ml.), recorded by the Cary 11MS re- cording spectrometer. Average recording time, 10 minutes f 1 minute. Solid line, spectrum obtained immediately (within 0.5 minute) after dissolving in sulfuric acid; broken line, 120 minutes after dissolving in acid.

absorption at 237 rnp. The material resisted crystallization, but the in- fra-red spectrum was identical with that of I prepared by Hayano and Dorfman (10). The other components of the hydrolysis could not be identified and probably are isomerization products. 6/?-Hydroxycortisol was isolated in crystalline form from Fractions 61 to 66 after rechromatog- raphy on a Hyflo Super-Cel column (100 X 2.5 cm.). Crystallization of the major fraction from acetone gave colorless needles (m.p. 225-233’) which proved to be identical with I isolated by the hydrolysis of I-ac. The absorption spectrum of I in sulfuric acid was identical with that of I-ac.

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588 STEROID METABOLISM. I

Steroids IIa and II&-Both of these steroids of unknown structure were isolated as their acetates from Zone II (Fig. l), and only steroid IIb was isolated from Fractions 49 to 52 (Fig. 2). Zone II consisted of a mixture of at least two reducing steroids having similar mobilities, which were difficult to separate. The mixture was acetylated and chromatographed on a Hyflo Super-Cel partition column with benzene as the mobile phase. Two major reducing steroid acetates were eluted, and these will be referred to in order of increasing mobility, steroids IIa-ac. and IIb-ac. Steroid IIa-ac. was crystallized from 95 per cent ethanol to afford colorless prisms which melted at 223-230” (the larger crystals melted at 236”). The ul- traviolet absorption spectrum in methanol had a maximum at 242 rnw (E::m. 384). The infra-red spectrum had the following bands: hydroxyl at 3550, ester carbonyl at 1749, C-20 carbonyl at 1730, conjugated car- bony1 at the unusually high frequency 1709, double bond at 1631, and acetate at 1230 and 1209 cm.-‘.

Steroid IIb-ac. crystallized in dense colorless needles which melted at 212-220”. The ultraviolet absorption spectrum in methanol had a maxi- mum at 238 rnp (E15m. 448). The infra-red spectrum had the following bands: hydroxyl at 3500, ester carbonyl at 1739, C-20 carbonyl at 1721, conjugated C-3 1669, A4 double bond at 1631, and acetate at 1232 cm.-‘.

The absorption spectra in sulfuric acid of steroids IIa-ac. and IIb-ac. are shown in Fig. 6. Both steroids gave a green fluorescence in sulfuric acid. Steroids IIa-ac. and IIb-ac. have not been identified. Since Zone IV (Fig. 1) was identified as 1 l@ ,17a, 2Oa, 21-tetrahydroxy-4-pregnen-3- one, it seems, in view of the structure-chromatographic mobility relation- ships (ll), that steroids IIa and IIb may be Czl0~ derivatives of cortisol.

The free steroid IIb was isolated from Fractions 49 to 52 (Fig. 2) after rechromatography on a Hyflo Super-Cel column. The major component crystallized readily by trituration with acetone, m.p. 234-238”. The in- fra-red spectrum had the following bands: hydroxyl at 3500, C-20 carbonyl at 1702, C-3 conjugated carbonyl at 1647, and A4 double bond at 1625 cm.-‘.

Acetylation yielded colorless dense needles (from ethanol) which were identical (melting point, infra-red analysis, spectrum in sulfuric acid) with steroid IIb-ac. described above. Steroid IIa was not found after column chromatography.

1 lp ,17ol, 20~~) 21 -Tetrahydroxy -4-pregnen-S-one (I V)-This steroid was isolated from Zone IV (Fig. 1) and Fractions 44 to 48 (Fig. 2). Zone IV only slightly reduced the blue tetrazolium reagent and gave a strong orange-red dinitrophenylhydrazone. The eluate of this zone resisted crystallization and was chromatographed on a Hyflo Super-Cel column. The material came off the column with benzene containing 35 per cent methylene chloride and crystallized after trituration with neohexane and

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S. BURSTEIN AND R. I. DORFMAN 589

acetone, m.p. 232-236”. The i&a-red spectrum of this material was identical with that of 1 I/?, 17~~) 20~~) 21-tetrahydroxy-4-pregnen-3-one

The spectrum in sulfuric acid of the isolated compound (m.p. 239-243’).’

1.5

Steroid II a

Steroid II b

wave length t& mp FIG. 6. Absorption spectra of steroids IIa-ac. and IIb-ac. in sulfuric acid (approxi-

mately 100 7 in 3 ml.), recorded by the Cary 11MS recording spectrometer. Average recording time, 10 minutes f 1 minute. Solid line, spectrum obtained within 0.5 minute after dissolving in sulfuric acid; broken line, 120 minutes after dissolving in acid.

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590 STEROID METABOLISM. I

IV was identical with that of the authentic sample (see Fig. 7). A transi- ent green fluorescence in sulfuric acid was observed. 1 l/l, 17~~) 20~1,21-Tet- rahydroxy-4-pregnen-3-one was isolated from Fractions 44 to 48 after

l-

fA,

H&70(, 20d,?l-Tatrahydvoxy -4-prayem-33-w

IlR,17d,20~,21-T~tv~hydroxy -4-prqnme-3,20-dione

22( ) 240 2&J 280 300 320 350 400 450 9% 550

Wd ve length lh tpl

10

FIG, 7. Absorption spectra of llp,17ar,2&~,21-tetrahydroxy-4-pregnen-3-one (ap- proximately 150 y in 3 ml.) and llfl,17o1,20~,21-tetrahydroxy-4-pregnen-3-one (ap- proximately 100 y in 3 ml.) in sulfuric acid, recorded by the Cary 1lMS recording spectrometer. Average recording time, 10 minutes f 1 minute. Solid line, spec- trum obtained within 0.5 minute after dissolving in sulfuric acid; broken line, 120 minutes after dissolving in acid.

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S. BURSTEIN AND R. I. DORFMAN 591

chromatography on a Hyflo Super-Cel column (100 X 2.5 cm.). Two major fractions were obtained which were identified as the 2Oa( and 200 isomers.

11/3,17a ,20/3,21 -Tetrahydroxy-$pregnen-S-one (V)-This steroid was iso- lated from Zone V (Fig. 1) and Fractions 44 to 48 (Fig. 2). Zone V gave the same color reactions as Zone IV, suggesting an intact A4-3-keto system with a reduced side chain. Paper chromatography of Zone V in the tolu- ene-propylene glycol system for 72 hours, followed by elution of the major steroid component and crystallization from acetone, resulted in crystalline material melting unsharply around 120”. The infra-red absorption spec- trum and the spectrum in sulfuric acid (Fig. 7) of this material were identi- cal with those of authentic 1 I/?, 17a, ZOP, 21-tetrahydroxy-4-pregnen-3-one monohydrate. A transient fluorescence in sulfuric acid was observed. The acetate prepared in the usual manner melted at 210-216’ (a change of crystal form from plates to needles occurred at 179”). The infra-red spec- trum of the diacetate was identical with that of an authentic sample of 2Op, 21-diacetoxy-llp , 17a-dihydroxy-4-pregnen-20-one.3 Because of the similarity of the infra-red spectra of steroids IV and V, identification of these materials was difficult by this means. However, the absorption spec- tra in sulfuric acid were distinctly different (Fig. 7).

Co&sol--Cortisol was isolated from Zone VII (Fig. 1) and Fractions 23 to 29 (Fig. 2). Repeated paper chromatography in the toluene-propylene glycol system of the eluate of Zone VII revealed only a single reducing zone which was identified as cortisol by melting point determinations and infra- red spectrometry (2).

Zones III, VI, and VIII and additional steroids in the overflow were pres- ent in small amounts and were all a,/3-unsaturated ketones as revealed by their ultraviolet absorption around 240 rnp. However, no conclusive identification of these zones was achieved. In addition, other fractions were eluted from the partition column, but were not identified with the exception of Fractions 107 to 110 which proved to be urea.

Determination of Cortisol Metabolites-By means of ultraviolet spectro- photometry and a quantitative paper chromatographic procedure described previously (2), the steroids in Zone I (GP-hydroxycortisol), Zone II (the mixture of steroids IIa and IIb), and Zone VII (cortisol) were quantita- tively determined after administration of single doses of 20, 40, and 80 mg. of cortisol to individual guinea pigs. The results are presented in Table I. When an animal was used for more than one dosage level, an interval of 1 week elapsed between feedings. The steroids in Zones I, II, and VII were excreted in the urine in concentrations of 0.5 f 0.1, 0.5 f 0.10, and 2.7 per cent f 0.3 per cent, respectively, of the 20 mg. of cortisol fed. The

3 Generously supplied by Professor T. Reichstein.

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592 STEROID METABOLISM. I

administration of 40 mg. of cortisol resulted in the following percentage con- version into these urinary steroids: 0.8 f 0.1, 0.4 f 0.1, and 1.9 f 0.4, respectively, and the administration of 80 mg. gave 0.9 f 0.1, 0.7 f 0.1, and 2.0 f 0.6, respectively. The variation of recoveries from one experi- ment to another was so great that one is not justified in concluding that the

TABLE I

Quantitative Determination of Steroids in Zone I (G&Hydroxycortisol), Zone II (Mixture of IIa and IIb), and Zone VIZ (Co&sol) by Ultraviolet

Spectrophotometry

Cortisol administered

- !

fw. 20

Mean f s.e.t..

40

Mean f s.e.. 320 f 30 144 f 27 748 f 122

80

Mean f se.. .

--

-_

.-

--

-

Guinea pig No. I- Zone I T

Zone II Zone VII

1 2 3 3 3 5 5 5 7 9

10 14

7 Y

90 50 160 170 50 80 10 68

240 50

50 50

220 100 109

50 110 120 110 170

Y

510 880 316 920 750 456 730 430 400 405 200 500

108 f 24 98 f 15 541 f 66

4 280 100 980 6 420 200 600 8 280 100 340

11 360 220 960 18 260 100 860

4 840 680 3460 6 520 440 1600 8 480 360 480

11 800 440 1240 18 800 680 1160

T Steroid recovered*

688 f 77 520 f 67 1588 z!z 502

* All values were calculated on the basis of the measured extinction coefficient of cortisol at X = 240 mp.

t Standard error.

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S. BURSTEIN AND R. I. DORFMAN 593

excretion of the reducing steroid is significantly affected by the dose of cortisol administered. Table II includes the weight distribution in the various fractions following column chromatography. Of the crude crystal- line ethyl acetate extract (145 mg.) applied to the column, 106 per cent was recovered in the 138 fractions collected (Fig. 2). A urine extract from the ten guinea pigs used prior to cortisol feeding weighed 20 mg. On the basis of crude crystalline weight (Table II), 4.6 per cent of the administered cortisol was recovered in the urine as such, 2.2 per cent as the 2oCr- and

TABLE II Chromatographic Fractionation of Extract of Guinea Pig Urine (146 Mg.) after Oral

Administration of 1 Gm. of Cortisol on Hyjlo Super-Ccl Column

Fraction No.

l-14 15-22 23-29 3043 44-48

49-52 6.3 53-57 5.3 58-60 0.9 61-66 17.7

67-106 21.0 107-110 4.6 111-138 3.6

T

Weight

mg.

7.0 9.4

46.4 10.4 21.5

Total 154.1

Major component

Cortisol

11@,17a,20a,21-Tetrahydroxy-4-pregnen-3- one

11,9,17~,20~,21-Tetrahydroxy-4-pregnen-3- one

Steroid IIb Unidentified ring A-saturated steroid

6&11~,17c~, 21-Tetrahydroxy-4-pregnene- 3,20-dione

Urea

20p-dihydro derivatives, 1.8 per cent as 6fl-hydroxycortisol, and 0.63 per cent as steroid IIb. A similar chromatographic pattern was obtained in a second experiment, yielding 3.7 per cent cortisol, 2.1 per cent of the ~OCY- and 20@dihydro derivatives, 1.5 per cent of the 6/?-hydroxycortisol, and 0.4 per cent of steroid IIb.

DISCUSSION

It has been found that, following the administration of cortisol to normal adult male guinea pigs, a series of metabolites was excreted in the urine. BP-Hydroxycortisol, 1 l/I, 17~~) 20~~) 21-tetrahydroxy-4-pregnen-3-one, and

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594 STEROID METABOLISM. I

lip, 17a!, ZOp, 21-tetrahydroxy-4-pregnen-3-one were isolated and identified as the major metabolites excreted in the free (unconjugated) form.

The isolation and identification of 6@hydroxycortisol establishes the first instance of 6/+hydroxylation in viva. The only 6-hydroxylated ste- roids thus far isolated from urine have been of the 6a! configuration from human pregnancy urine (12). 6@Hydroxylation has been demonstrated by the incubation of 11-deoxycorticosterone with hog adrenal mince (13), with homogenates of cow corpus luteum (14), and by the perfusion of progesterone through beef adrenals.4 Recently, 6@-hydroxylation of I l-de- oxycorticosterone, testosterone, and A4-androstene-3, 17-dione has been shown (by paper chromatography and color reactions) to occur in perfused rat liver (15). An efficient Go-hydroxylating system has been shown to exist in microbiological preparations of R&opus urrhizus (16).

The biological significance of the 6/3-hydroxylation in the guinea pig is not apparent. The isolation of 6P-hydroxycortisol from the urine of nor- mal and scorbutic guinea pigs6 shows that 6P-hydroxylation in this animal occurs also under physiological conditions. 6/3-Hydroxylation in viva is not unique for the guinea pig, since 6@hydroxycortisol has now been isolated from human urine after administration of cortisol and from late human pregnancy urine (17).

Three minor zones were found in Zone I, less mobile than 6@-hydroxy- cortisol. One of the steroids in these zones may be the 6or isomer. How- ever, the possibility that these zones may be artifacts should be considered in view of the isomerizations found by Herzig and Ehrenstein (8).

Steroids IIa and IIb seem to be (in respect to mobility) A4-3-keto CZ~OS steroids, the type and position of the additional oxygen being unknown, which would indicate that in addition to the G/3-hydroxylation another process for the introduction of an additional oxygen function is operating. Steroid Ha has been also isolated from the urine of normal and scorbutic guinea pigs.5

Another metabolic process established in this study is the reduction of cortisol to both the 20~ and 20p-dihydro derivatives.

SUMMARY

After administration of cortisol to guinea pigs, the following steroids were isolated and identified as the major metabolites present in the urine in the unconjugated form, in addition to unchanged cortisol: Sp, 11/3,17a, - 21-tetrahydroxy-4-pregnene-3,20-dione (I), 1 lp ,17a, 20~~) 21-tetrahydroxy- 4-pregnen-3-one (IV), and llp,17a, 20/3,21-tetrahydroxy-4-pregnen-3-one (V). Two unidentified steroids (IIa and IIb) were also isolated and seem

4 Levy, H., unpublished data. 6 Burstein, S., Dorfman, R. I., and Nadel, E. M., to be published.

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S. BURSTEIN AND R. I. DORFMAN 595

to be (from mobility considerations) A4-3-keto C&O6 derivatives of cortisol. Steroids I, IV + V, IIb, and cortisol, on the basis of crude crystalline weight determined by column partition chromatography, were excreted in concen- trations of 1.77, 2.15, 0.63, and 4.64 per cent, respectively, of the fed corti- sol.

Thanks are due to Dr. Frank Ungar for help in the preparation of the manuscript.

BIBLIOGRAPHY

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(1951). 5. Burstein, S., Savard, K., and Dorfman, R. I., Endocrinology, 63, 88 (1953). 6. Butt, W. R., Morris, P., Morris, C. J. 0. R., and Williams, D. C., Biochem. J.,

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and Paul R. Skogstromthe technical assistance of Arline M. Tillotson Shlomo Burstein, Ralph I. Dorfman and With

IN VIVOPIGS: I. METABOLISM OF CORTISOL STEROID METABOLISM IN GUINEA

1955, 213:581-595.J. Biol. Chem. 

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