- Email: [email protected]
6β-hydroxycortisol metabolism in man
529
6S-HYDROXYCORTISOL METABOLISM IN MAN. Ross Dixon? and G.W. Pennington. Sheffield and Region Endocrine Investigation Centre, Jessop Hospital for Women, Sheffield 10, England.
Received January 8, 1969
ABSTRACT
6f3-hydroxycortisol (6S-OH-F) metabolism was studied by i.v. administration of 1,2-3H-6S-OH-F to a normal male. Of the excreted radioactivity, 80% was found in the unconjugated fraction and there was no evidence for conjugation with either glucuronic acid or sulfuric acid. The principal urinary steroid was 66-OH-F and constituted about half the radioactivity present. Reduction of the 20-ketone to yield the 2OSdihydro derivative of 6@-OH-F and oxidation of the llS-hydroxyl group to give GS-hydroxycortisone (6B-OH-E) proved to be minor metabolic transformations. The presence of several other, as yet unidentified, minor urinary metabolites of 6S-OH-F has also been noted.
It has previously been suggested by Dixon and Pennington (1) that two separate biochemical pathways are operative in the transformation of cortisol to 20-dihydro-6S-OH-F which has been isolated from urine (2). The first involves the transformation of cortisol to 6B-OH-F which is then reduced at the C-20 position.
Alternatively, cortisol may be re-
duced at C-20 to give 20-dihydro-F and, subsequently, 6$-hydroxylated. Intravenous administration of radioactive 2OB-dihydro-F has demonstrated that the latter pathway is operative in vivo but that 2OS-dihydro-6S-OH-F is not solely derived from this circulating precursor (1). The present study was primarily carried out in order to investigate the possibility that G&-OH-F, which has recently been shown to be a secretory product of the normal adrenal (3), might also serve as a circulating precursor of 20-dihydro-6S-OH-F.
STEROIDS
530
13:4
The metabolism of 68-OH-F was first studied by Burstein et al. (3) and these workers have reported that 59% of the administered radioactive dose was excreted in an unconjugated form within 48 hours.
No attempt was
apparently made, however, to determine the total excretion of radioactivity and/or to what extent, if any, there was further metabolism and conjugation with either glucuronic or sulfuric acid. It was, therefore, of interest in the present study to investigate the distribution of any other metabolites of @-OH-F
in urine and to observe if
6H-OH-F represented an end product in the metabolism of cortisol prior to excretion.
MATERIALS ANDMETHODS
Solvent purification, mass spectrometry and measurement of radioactivity were carried out as described in a previous publication (1). Celite columns were prepared as described by Siiteri (4) and thin layer chromatography was performed on Silica Gel254 (E. Merck AG). Paper chromatograms were obtained on Whatman No, 1 paper and, in certain instances, impregnated with boric acid as described by Schneider and Lewbart (5). The chromatographic systems are listed in Table I. Preparation of 1,2-3H-6B-hydroxycortisol. 1 mc of 1,2-3H-cortisol (specific activity 5 mc/mg) was administered intravenously to a volunteer. Urine was collected for 24 hours and extracted with ethyl acetate according to the procedure of Frantz et al. (6). The extract was chromatographed on paper in System P-4 for 5 hours and the zone coincident with an adjacent marker of 68-OH-F eluted with methanol. 6$-OH-F is readily separated from its 6u-epimer in this system. The eluted material was rechromatographed in System P-l and a radioactive scan of the chromatogram showed a single symmetrical peak coincident with 6f3-OH-F. Following elution, the radioactive material was acetylated and further chromatographed on paper in System P-5. A single radioactive UV absorbing band coincident with a marker of 6$-OH-F diacetate was found which had a specific activity of 6960 cpm/pg expressed as BS-OH-F when quantitated by blue-tetrazolium reduction. Following hydrolysis under nitrogen for 15 hours in lml of methanol containing 0.2 ml of 1% aqueous sodium bicarbonate, the product was chromatographed on paper in System P-2 where it migrated as GB-OH-F and showed only a single radioactive peak. Aliquots of the material behaved in a similar manner to 6+OH-F following paper chromatography in Systems P-l and P-3 exhibiting, in each instance, a single soda-fluorescent and blue-tetrazolium spot. The specific activity of the purified 1,2-3H6B-hydroxycortisol was 7,100 cpm/pg.
531
STEROIDS
April 1969
TABLE
I
CHROMATOGRAPHIC SYSTEMS. COMPONENTS
SUPPORT Celite
C-l
Isooctane:t-butanol:l M ammonia
2:5:5
c-2
Isooctane:t-butanol:l M ammonia
3:5:5
c-3
Chloroform:ethyl acetate:methanol:water
9:1:5:5
c-4
Isooctane:ethyl acetate:methanol:water
3:5:1:3
c-5
Benzene:ethyl acetate:methanol:water
3:1:1:2
C-6
Benzene:ethyl acetate:methanol:water
2:1:1:2
C-7
Isooctane:t-butanol:methanol:water
6:4:1:4
c-8
Hexane:ethyl acetate:methanol:water
4:3:3:2
P-l
Benzene:ethyl acetate:methanol:water
7:3:10:10
P-2
Benzene:n-butanol:methanol:water
9:1:3:3
P-3
Benzene:+butanol:vater
70~43~86
P-4
Benzene:acetone:water
2:1:2
P-5
Toluene:light petroleum (40-60"):methanol:water
5:5:4:1
P-6
Benzene:methanol:water
2:l:l
TLC-l
Chloroform:ethanol:water
80:20:1
TLC-2
Ether:ethanol
9:l
TLC-3
Ether
Administration of tracer. A dose of 5.1 x lo6 cpm (715 ug) of 1,2-3H-6B-hgdroxycortisol was administered intravenously in 10 ml of 10% ethanol in isotonic saline to a 30 year old endocrinologically normal male. Urine was collected in separate daily batches and the radioactive content of each collection assayed (Table II). A preliminary study on the amount of radioactivity extractable before and after hydrolysis with S-glucuronidase and sulfatase. Extraction with ethyl acetate. 50 ml of the first 24-hour urine collection was extracted once with 100 ml of ethyl acetate following the addition of 20% (w/v) NaCl. The extract was washed twice with 0.1 N NaOH containing 30% (w/v> NaCl followed by a single wash with saturated NaCl. Solvent evaporation yielded a residue containing 52,000 cpm (Table II).
(1180 ml)
(1320 ml)
(1240 ml)
Day 1.
Day 2.
Day 3.
x lo5
1.01 x 106 1.4 5.7
9.59 x 105 8.7
5.25 x lo4
Tetrahydrofuran extraction (Free Fraction)
Solvolyzable Fraction
6B-OH-F
6E-OH-E
20f3-dihydro-6S-OH-F
(b) % of the total radioactivity excreted in Day 1.
3
5
57
3.3
80
59
70
% Urine(b)
1.2
34
% Dose
(a) Counts are corrected for the extraction of all the Day 1 urine specimen.
x lo4
x 104
x 106
1.23 x l06(a)
Enzyme hydrolysis
Not detected
0.6
1.74 x 106
5.1 x 106
SP!!!
Ethyl acetate extraction
Day 1.
Urine
Dose
II
METABOLISM OF 1,2-3H-6S-HYDROXYCORTISOL
TABLE
u-l
N
w
April
1969
533
STEROIDS
Enzyme hydrolysis and extraction. An additional 50 ml portion of the urine was adjusted to pH 5 and made 0.2 M with sodium acetate buffer (pH 5). f3-glucuronidase (Ketodase) and sulfatase (Helicase) were added to give a final concentration of 500 and 2500 units/ml, respectively. The solution was incubated at 37' for 2 days after which the pH was adjusted to 7.5 and extracted with ethyl acetate as previously described. A single wash with 10% (w/v) Na2C03 in 20% (w/v) NaClwas performed prior to washing with NaOH. Solvent evaporation yielded a residue containing 43,400 cpm (Table II). Isolation of the urinary metabolites of 1,2-3H-68-hydroxycortisol. To of NaCl organic ately 2
800 ml of the first 24-hour urine and the solution extracted with 1 extract was evaporated to yield a g and contained 950,000 cpm (Free
specimen, at pH 6, was added 200 g liter of tetrahydrofuran. The brown oil which weighed approximFraction).
The remaining aqueous phase was completely saturated with NaCl and acidified to pH 1 with 50% sulfuric acid. The solution was extracted with 1 liter of tetrahydrofuran after which the organic extract was filtered and 0.9 ml of 60% perchloric acid added. The clear yellow solution was solvolyzed at 37" for 18 hours (7). After addition of 2 ml of pyridine, the solvent was evaporated and the resulting dark oil dissolved in 50 ml of 20% (w/v) NaCl. The pH was adjusted to 7 and the solution extracted three times with two volumes of ethyl acetate. The combined extracts were washed, as previously described, with alkali and saline and the solvent evaporated to yield an oily residue containing 39,000 cpm (Solvolyzable Fraction). Chromatography of Free Fraction. The crude extract was chromatographed on 120 g Celite [holdback volume (HBV) = 180 ml] using Solvent System C-2. Four zones of radioactivity were eluted in HBV O-l (Zone I), HBV l-2.5 (Zone II), HBV 2.5-4 (Zone III) and HBV 4-6 (Zone IV). Zone I. Isolation of 6B-OH-E. Zone I, which contained 161,000 cpm, was chromatographed on 44g Celite with System C-5. After collecting 5 HBV, the mobile phase of System C-6 was used for elution and an additional 2 HBV collected. Four radioactive components separated and were eluted in HBV 1, l-2, 2-2.8 and 3-4.5. The material (57,800 cpm),eluted in HBV 2-2.8, behaved as 68-OH-E on both paper and Thin Layer Chromatography (TLC) with Systems P-l and TLC1, respectively. The metabolite exhibited a maximum absorption at 232 rnp in methanol. The specific activity, as determined by its UV absorption and blue tetrazolium reduction against standard 6S-OH-E, was 1383 and 1380 cpm/pg, respectively. Acetylation and TLC, yielded a single UV absorbing spot with the same mobility as 6S-OH-E &acetate and had a specific activity of 1364 cpm/ g expressed as 6S-OH-E. The identity of the other metabolites eluted from the Celite column could not be established. Zone II. Isolation of 6B-OH-F. The pigmented residue,which weighed 87 mg and contained 795,000 cpm, was rechromatographed on 66 g Celite using System C-4. A large symmetrical peak of radioactivity was eluted
534
STEROIDS
13:4
in HBV 3-5. Solvent evaporation yielded an amorphous white residue containing 673,000 cpm. Crystallization from acetone-hexane gave 2.5 mg of fine needles, m.p. 280-284", h 212 and 270 mu. The infxared spectrum suggested that the compound wag%on-steroidal in nature. The mother liquor from the first crystallization was rechromatographed on 16 g Celite using System C-6 and a single symmetrical peak of radioactivity was eluted in HBV 4-5. 6@-OH-F is normally eluted in these fractions with System C-6. The almost colorless residue, obtained upon solvent evaporation, contained 610,000 cpm. Paper chromatography for 15 hours in System P-2 gave a single radioactive peak 20 em from the origin and coincident with a marker of 6S-OH-F. Examination of the chromatogram under UV light (254 mu) showed the presence of fluorescent contaminants in the radioactive‘on:&e but theaa were successfully removed by TLC with System TLC-Z. The material exhibited a maximum absorption at 237 mu in methanol and had specific activities of 2610 and 2540 cpm/pg when determined by its UV absorption and bluetetrazolium reduction against standard 68-OH-F, Apart from a broader carbony1 band at 1665 cm-l, the infrared spectrum was identical with that of 68-OH-F,
Zone III. Isolation of 20@-Dihydro-68-OH-F. The pale yellow amorphous residue, which weighed 29 mg and contained 107,000 cpm, was rechromatographed on 33 g Celite using System C-3. Zone III separated into three radioactive peaks which were eluted in HBV 2-3, 3.5-5 and 5-6. The material eluted in HBV 2-3 (35,450 cpm) gave a single radioactive peak following paper chromatography in System P-2 for 17 hours. The material was rechromatographed in the same system on boric acid impregnated paper for 18 hours. Again, a single radioactive peak 28 cm from the origin was found. Adjacently run markers of ZOa-dihydro-68-OH-F and the 20B-epimer migrated 13 and 28 cm, respectively. An aliquot of the radioactive metabolite was oxidized with sodium bismuthate and the product chromatographed on paper in System P-6 for 15 hours. A single sodafluorescent spot, exactly coincident with a marker of 6B,lli3-dihydroxyandrost-4-ene-3,17-dione was observed. The remainder of the material was acetylated and chromatographed on 16 g of Celite using System C-8. A single symmetrical peak of radioactivity was eluted in HBV 4-6. Paper chromatography of the combined fractions in System P-5 for 12 hours showed a single ultraviolet absorbing band 17 cm from the origin and coincident with a marker of ZOB-dihydro-GB-triacetate. The acetate was eluted, further purified by TLC using System TLC-3 and found to exhibit a maximum absorption at 237 mu. The mass spectrum was identical with that of ZOS-dihydro-68-OH-F triacetate and showed major peaks at m/e 464(M-42), 446(M-60), 428@-60-18), 386, 368, 240, 225 and 173. The remainder of the acetate was oxidized for 10 minutes with 0.5% chromium trioxide in 95% acetic acid and the product chromatographed on silica gel with System TLC-3 where a single UV absorbing spot, coincident with 20S-dihydro-68OH-E triacetate was observed. The eluted material showed a maxims absorption at 234 mu while authentic 20S-dihydro-6S-OH-E similarly absorbed at this wavelength. The specific activities of the isolated 208-dihydro6B-OH-F, on the basis of its UV absorption as ZOB-dihydro-6B-OH-F triacetate and ZOS-dihydro-E triacetate, were 950 and 900 cpm/ug, respect-ively.
535
STEROIDS
April1969
The metabolites eluted in HBV 3.5-5 (27,950 cpm) and S-6 (23,200 cpm) each showed single symmetrical radioactive peaks at 16 cm from the origin following paper chromatography in System P-2 for 21 hours. Following acetylation, both acetates when chromatographed together in System P-5, gave single radioactive peaks with the same Rf value of 0.65. No ultraviolet absorption could be detected in either radioactive zone. An adjacently run marker of 208-dihydro-6$-08-F triacetate.had an Rf value of 0.15, No meaningful mass spectra were obtained from either acetate. Zone IV. The residue which contained 41,500 cpm, gave a single radioactive peak in HBV 1.5-2 following chromatography on 22 g Celite with System C-l. Repeated attempts to remove contaminating material by column chromatography and TLC were unsuccessful. Solvolyzable Fraction. Due to the small amount of radioactivity in relation to the large amount of contaminating pigments and the likelihood of multiple artifacts from the solvolytic procedure, no further analysis of this fraction was undertaken.
RESULTS AND DISCUSSION
Following the intravenous administration of l,2-3H-68-hydroxycertisol to an endocrinologically normal male, 34% of the injected dose was excreted in the urine during the first 24 hours (Table II).
Only an
additional 1.2% could be detected in the 48-hour collection and none in the 72-hour sample.
This excretion of radioactivity would appear to be
somewhat lower than the excretion encountered by Burstein et al. (3) who were
able to extract
collection.
59% of the injected dose from the 48-hour urine
These workers did not, however, measure the total amount of
radioactivity excreted.
During the present study, it was found possible
to extract 80% of the total urinary radioactivity by the use of tetrahydrofuran (Table II). remaining 20%.
It was not found possible to account for the
Hydrolysis of the urine with both S-glucuronidase and
sulfatase and subsequent ethyl acetate extraction did not yield any further amounts of radioactivity than were found in unhydrolyzed urine (Table II).
It was of interest to note that the amount of radioactivity
536
STEROIDS
13:4
extracted following hydrolysis was, in fact, less than that obtained without hydrolysis.
A possible explanation is the extra alkali wash to which
the hydrolyzed extract was subjected.
Although no significant amounts of
conjugated metabolites appeared to be present in the urine, extraction with tetrahydrofuran yielded 10% more radioactivity than ethyl acetate extsaction.
This can be explained on the assumption that some of the more
polar unidentified metabolites were not adequately extracted with ethyl acetate and a portion of the metabolites which were extracted were destroyed during alkali washing. Extraction of the urine at pH 1 with tetrahydrofuran, following previous extraction with the same solvent at pH 6, did not yield any further significant amounts of radioactivity after solvolysis with perchloric acid. This solvolytic procedure readily splits C-21 ester sulfates (7).
It can-
not be assumed, however, from this latter observation, that sulfated metabolites of 66-OH-F were not present.
Such metabolites would be extreme-
ly polar in nature and possibly not extracted by tetrahydrofuran at pH 1. In contrast to the in vivo metabolism of cortisol in the normal subject where approximately 70-80% of the administered dose is excreted in the urine in 24 hours, only 34% was excreted after the administration of 6@-OH-F.
It is possible that a sizeable portion of the remaining radio-
activity was excreted in the feces.
It is also of interest to note that
the major portion (57%) of the excreted radioactivity was found to be 6B-OH-F, whereas cortisol is primarily metabolized to tetrahydrocortisol and tetrahydrocortisone and followed by conjugation with glucuronic acid. The present study does demonstrate, however, that 68-OH-F, apart from its apparent lack of conjugated metabolites, does undergo several quantitatively minor chemical transformations.
These include oxidation of the
April 1969
STEROIDS
118-hydroxyl group and reduction of the C-20 carbonyl group to the 208configuration. Comparison of the specific activity of urinary 68-OH-F with those of the urinary 20B-dihydro-68-OH-F and 6#3-OH-E has indicated that 36% of urinary 2GB-dihydro-6%-08-F and 54% of urinary 68-OH-E were derived from circulating 6@-OH-F.
These results support the earlier suggestion that
two biochemical pathways are operative for the formation of 20B-dihydro-6SOM-F from cortisol:
the first involving 68-hydroxylation of 208-dihydro-
cortisol and the second, 20+reduction
of 68-OH-F.
t Present Address: Department of Obstetrics and Gynecology, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research Council of Great Britain. The authors are grateful to Mr. Stephen Jones, Department of Chemistry, Sheffield University, for determining the mass spectra. The secretarial assistance of Mrs. Selma J. Nagy, in the preparation of this manuscript, is also appreciated.
1.
Dixon, R. and Pennington, G.W., STEROIDS 12, 423 (1968).
2.
Dixon, R. and Pennington, G,W., J. ENDOCRINOL. 34, 281 (1966).
3.
Burstein, S., Kimball, H-L., Klaiber, E.L. and Gut, M., J. CLIN. ENDOCR. 27_, 491 (1967).
4.
Siiteri, P.K., STEROIDS 2, 687 (1963).
5.
Schneider, J.J. and Lewbart, M., TETRAHEDRON 20, 943 (1964).
6.
Frantz, A.G., Katz, F.H. and Jailer, J.W., J. CLIN. ENDOCR. 2, (1961).
1290
538
7.
STEROIDS
13:4
Burstein, S. and Lieberman, S., J. BIOL. CHEM. 233. 331 (1958).
GLOSSARY
The systematic names of steroids, identified in the text by trivial names or abbreviations, are: 6B-OH-F = 6B-hydroxycortisol = 68,11@,17cr,21-tetrahydroxypregn-4-ene-3,20dione. 2Oa(@)-dihydro-F = 20a(B)-dihydrocortisol = llB,17a,20a(B), 2l_tetrahydroxypregn-4-ene-3-one. 20a(@)-dihydro-68-OH-F = 68,118,17a,20a(8), 21-pentahydroxypregn-4-ene-3one. 6B-OH-E = 6@-hydroxycortisone = 68,17a,21-trihydroxypregn-4-ene-3,11,20trione.
6S-HYDROXYCORTISOL METABOLISM IN MAN. Ross Dixon? and G.W. Pennington. Sheffield and Region Endocrine Investigation Centre, Jessop Hospital for Women, Sheffield 10, England.
Received January 8, 1969
ABSTRACT
6f3-hydroxycortisol (6S-OH-F) metabolism was studied by i.v. administration of 1,2-3H-6S-OH-F to a normal male. Of the excreted radioactivity, 80% was found in the unconjugated fraction and there was no evidence for conjugation with either glucuronic acid or sulfuric acid. The principal urinary steroid was 66-OH-F and constituted about half the radioactivity present. Reduction of the 20-ketone to yield the 2OSdihydro derivative of 6@-OH-F and oxidation of the llS-hydroxyl group to give GS-hydroxycortisone (6B-OH-E) proved to be minor metabolic transformations. The presence of several other, as yet unidentified, minor urinary metabolites of 6S-OH-F has also been noted.
It has previously been suggested by Dixon and Pennington (1) that two separate biochemical pathways are operative in the transformation of cortisol to 20-dihydro-6S-OH-F which has been isolated from urine (2). The first involves the transformation of cortisol to 6B-OH-F which is then reduced at the C-20 position.
Alternatively, cortisol may be re-
duced at C-20 to give 20-dihydro-F and, subsequently, 6$-hydroxylated. Intravenous administration of radioactive 2OB-dihydro-F has demonstrated that the latter pathway is operative in vivo but that 2OS-dihydro-6S-OH-F is not solely derived from this circulating precursor (1). The present study was primarily carried out in order to investigate the possibility that G&-OH-F, which has recently been shown to be a secretory product of the normal adrenal (3), might also serve as a circulating precursor of 20-dihydro-6S-OH-F.
STEROIDS
530
13:4
The metabolism of 68-OH-F was first studied by Burstein et al. (3) and these workers have reported that 59% of the administered radioactive dose was excreted in an unconjugated form within 48 hours.
No attempt was
apparently made, however, to determine the total excretion of radioactivity and/or to what extent, if any, there was further metabolism and conjugation with either glucuronic or sulfuric acid. It was, therefore, of interest in the present study to investigate the distribution of any other metabolites of @-OH-F
in urine and to observe if
6H-OH-F represented an end product in the metabolism of cortisol prior to excretion.
MATERIALS ANDMETHODS
Solvent purification, mass spectrometry and measurement of radioactivity were carried out as described in a previous publication (1). Celite columns were prepared as described by Siiteri (4) and thin layer chromatography was performed on Silica Gel254 (E. Merck AG). Paper chromatograms were obtained on Whatman No, 1 paper and, in certain instances, impregnated with boric acid as described by Schneider and Lewbart (5). The chromatographic systems are listed in Table I. Preparation of 1,2-3H-6B-hydroxycortisol. 1 mc of 1,2-3H-cortisol (specific activity 5 mc/mg) was administered intravenously to a volunteer. Urine was collected for 24 hours and extracted with ethyl acetate according to the procedure of Frantz et al. (6). The extract was chromatographed on paper in System P-4 for 5 hours and the zone coincident with an adjacent marker of 68-OH-F eluted with methanol. 6$-OH-F is readily separated from its 6u-epimer in this system. The eluted material was rechromatographed in System P-l and a radioactive scan of the chromatogram showed a single symmetrical peak coincident with 6f3-OH-F. Following elution, the radioactive material was acetylated and further chromatographed on paper in System P-5. A single radioactive UV absorbing band coincident with a marker of 6$-OH-F diacetate was found which had a specific activity of 6960 cpm/pg expressed as BS-OH-F when quantitated by blue-tetrazolium reduction. Following hydrolysis under nitrogen for 15 hours in lml of methanol containing 0.2 ml of 1% aqueous sodium bicarbonate, the product was chromatographed on paper in System P-2 where it migrated as GB-OH-F and showed only a single radioactive peak. Aliquots of the material behaved in a similar manner to 6+OH-F following paper chromatography in Systems P-l and P-3 exhibiting, in each instance, a single soda-fluorescent and blue-tetrazolium spot. The specific activity of the purified 1,2-3H6B-hydroxycortisol was 7,100 cpm/pg.
531
STEROIDS
April 1969
TABLE
I
CHROMATOGRAPHIC SYSTEMS. COMPONENTS
SUPPORT Celite
C-l
Isooctane:t-butanol:l M ammonia
2:5:5
c-2
Isooctane:t-butanol:l M ammonia
3:5:5
c-3
Chloroform:ethyl acetate:methanol:water
9:1:5:5
c-4
Isooctane:ethyl acetate:methanol:water
3:5:1:3
c-5
Benzene:ethyl acetate:methanol:water
3:1:1:2
C-6
Benzene:ethyl acetate:methanol:water
2:1:1:2
C-7
Isooctane:t-butanol:methanol:water
6:4:1:4
c-8
Hexane:ethyl acetate:methanol:water
4:3:3:2
P-l
Benzene:ethyl acetate:methanol:water
7:3:10:10
P-2
Benzene:n-butanol:methanol:water
9:1:3:3
P-3
Benzene:+butanol:vater
70~43~86
P-4
Benzene:acetone:water
2:1:2
P-5
Toluene:light petroleum (40-60"):methanol:water
5:5:4:1
P-6
Benzene:methanol:water
2:l:l
TLC-l
Chloroform:ethanol:water
80:20:1
TLC-2
Ether:ethanol
9:l
TLC-3
Ether
Administration of tracer. A dose of 5.1 x lo6 cpm (715 ug) of 1,2-3H-6B-hgdroxycortisol was administered intravenously in 10 ml of 10% ethanol in isotonic saline to a 30 year old endocrinologically normal male. Urine was collected in separate daily batches and the radioactive content of each collection assayed (Table II). A preliminary study on the amount of radioactivity extractable before and after hydrolysis with S-glucuronidase and sulfatase. Extraction with ethyl acetate. 50 ml of the first 24-hour urine collection was extracted once with 100 ml of ethyl acetate following the addition of 20% (w/v) NaCl. The extract was washed twice with 0.1 N NaOH containing 30% (w/v> NaCl followed by a single wash with saturated NaCl. Solvent evaporation yielded a residue containing 52,000 cpm (Table II).
(1180 ml)
(1320 ml)
(1240 ml)
Day 1.
Day 2.
Day 3.
x lo5
1.01 x 106 1.4 5.7
9.59 x 105 8.7
5.25 x lo4
Tetrahydrofuran extraction (Free Fraction)
Solvolyzable Fraction
6B-OH-F
6E-OH-E
20f3-dihydro-6S-OH-F
(b) % of the total radioactivity excreted in Day 1.
3
5
57
3.3
80
59
70
% Urine(b)
1.2
34
% Dose
(a) Counts are corrected for the extraction of all the Day 1 urine specimen.
x lo4
x 104
x 106
1.23 x l06(a)
Enzyme hydrolysis
Not detected
0.6
1.74 x 106
5.1 x 106
SP!!!
Ethyl acetate extraction
Day 1.
Urine
Dose
II
METABOLISM OF 1,2-3H-6S-HYDROXYCORTISOL
TABLE
u-l
N
w
April
1969
533
STEROIDS
Enzyme hydrolysis and extraction. An additional 50 ml portion of the urine was adjusted to pH 5 and made 0.2 M with sodium acetate buffer (pH 5). f3-glucuronidase (Ketodase) and sulfatase (Helicase) were added to give a final concentration of 500 and 2500 units/ml, respectively. The solution was incubated at 37' for 2 days after which the pH was adjusted to 7.5 and extracted with ethyl acetate as previously described. A single wash with 10% (w/v) Na2C03 in 20% (w/v) NaClwas performed prior to washing with NaOH. Solvent evaporation yielded a residue containing 43,400 cpm (Table II). Isolation of the urinary metabolites of 1,2-3H-68-hydroxycortisol. To of NaCl organic ately 2
800 ml of the first 24-hour urine and the solution extracted with 1 extract was evaporated to yield a g and contained 950,000 cpm (Free
specimen, at pH 6, was added 200 g liter of tetrahydrofuran. The brown oil which weighed approximFraction).
The remaining aqueous phase was completely saturated with NaCl and acidified to pH 1 with 50% sulfuric acid. The solution was extracted with 1 liter of tetrahydrofuran after which the organic extract was filtered and 0.9 ml of 60% perchloric acid added. The clear yellow solution was solvolyzed at 37" for 18 hours (7). After addition of 2 ml of pyridine, the solvent was evaporated and the resulting dark oil dissolved in 50 ml of 20% (w/v) NaCl. The pH was adjusted to 7 and the solution extracted three times with two volumes of ethyl acetate. The combined extracts were washed, as previously described, with alkali and saline and the solvent evaporated to yield an oily residue containing 39,000 cpm (Solvolyzable Fraction). Chromatography of Free Fraction. The crude extract was chromatographed on 120 g Celite [holdback volume (HBV) = 180 ml] using Solvent System C-2. Four zones of radioactivity were eluted in HBV O-l (Zone I), HBV l-2.5 (Zone II), HBV 2.5-4 (Zone III) and HBV 4-6 (Zone IV). Zone I. Isolation of 6B-OH-E. Zone I, which contained 161,000 cpm, was chromatographed on 44g Celite with System C-5. After collecting 5 HBV, the mobile phase of System C-6 was used for elution and an additional 2 HBV collected. Four radioactive components separated and were eluted in HBV 1, l-2, 2-2.8 and 3-4.5. The material (57,800 cpm),eluted in HBV 2-2.8, behaved as 68-OH-E on both paper and Thin Layer Chromatography (TLC) with Systems P-l and TLC1, respectively. The metabolite exhibited a maximum absorption at 232 rnp in methanol. The specific activity, as determined by its UV absorption and blue tetrazolium reduction against standard 6S-OH-E, was 1383 and 1380 cpm/pg, respectively. Acetylation and TLC, yielded a single UV absorbing spot with the same mobility as 6S-OH-E &acetate and had a specific activity of 1364 cpm/ g expressed as 6S-OH-E. The identity of the other metabolites eluted from the Celite column could not be established. Zone II. Isolation of 6B-OH-F. The pigmented residue,which weighed 87 mg and contained 795,000 cpm, was rechromatographed on 66 g Celite using System C-4. A large symmetrical peak of radioactivity was eluted
534
STEROIDS
13:4
in HBV 3-5. Solvent evaporation yielded an amorphous white residue containing 673,000 cpm. Crystallization from acetone-hexane gave 2.5 mg of fine needles, m.p. 280-284", h 212 and 270 mu. The infxared spectrum suggested that the compound wag%on-steroidal in nature. The mother liquor from the first crystallization was rechromatographed on 16 g Celite using System C-6 and a single symmetrical peak of radioactivity was eluted in HBV 4-5. 6@-OH-F is normally eluted in these fractions with System C-6. The almost colorless residue, obtained upon solvent evaporation, contained 610,000 cpm. Paper chromatography for 15 hours in System P-2 gave a single radioactive peak 20 em from the origin and coincident with a marker of 6S-OH-F. Examination of the chromatogram under UV light (254 mu) showed the presence of fluorescent contaminants in the radioactive‘on:&e but theaa were successfully removed by TLC with System TLC-Z. The material exhibited a maximum absorption at 237 mu in methanol and had specific activities of 2610 and 2540 cpm/pg when determined by its UV absorption and bluetetrazolium reduction against standard 68-OH-F, Apart from a broader carbony1 band at 1665 cm-l, the infrared spectrum was identical with that of 68-OH-F,
Zone III. Isolation of 20@-Dihydro-68-OH-F. The pale yellow amorphous residue, which weighed 29 mg and contained 107,000 cpm, was rechromatographed on 33 g Celite using System C-3. Zone III separated into three radioactive peaks which were eluted in HBV 2-3, 3.5-5 and 5-6. The material eluted in HBV 2-3 (35,450 cpm) gave a single radioactive peak following paper chromatography in System P-2 for 17 hours. The material was rechromatographed in the same system on boric acid impregnated paper for 18 hours. Again, a single radioactive peak 28 cm from the origin was found. Adjacently run markers of ZOa-dihydro-68-OH-F and the 20B-epimer migrated 13 and 28 cm, respectively. An aliquot of the radioactive metabolite was oxidized with sodium bismuthate and the product chromatographed on paper in System P-6 for 15 hours. A single sodafluorescent spot, exactly coincident with a marker of 6B,lli3-dihydroxyandrost-4-ene-3,17-dione was observed. The remainder of the material was acetylated and chromatographed on 16 g of Celite using System C-8. A single symmetrical peak of radioactivity was eluted in HBV 4-6. Paper chromatography of the combined fractions in System P-5 for 12 hours showed a single ultraviolet absorbing band 17 cm from the origin and coincident with a marker of ZOB-dihydro-GB-triacetate. The acetate was eluted, further purified by TLC using System TLC-3 and found to exhibit a maximum absorption at 237 mu. The mass spectrum was identical with that of ZOS-dihydro-68-OH-F triacetate and showed major peaks at m/e 464(M-42), 446(M-60), 428@-60-18), 386, 368, 240, 225 and 173. The remainder of the acetate was oxidized for 10 minutes with 0.5% chromium trioxide in 95% acetic acid and the product chromatographed on silica gel with System TLC-3 where a single UV absorbing spot, coincident with 20S-dihydro-68OH-E triacetate was observed. The eluted material showed a maxims absorption at 234 mu while authentic 20S-dihydro-6S-OH-E similarly absorbed at this wavelength. The specific activities of the isolated 208-dihydro6B-OH-F, on the basis of its UV absorption as ZOB-dihydro-6B-OH-F triacetate and ZOS-dihydro-E triacetate, were 950 and 900 cpm/ug, respect-ively.
535
STEROIDS
April1969
The metabolites eluted in HBV 3.5-5 (27,950 cpm) and S-6 (23,200 cpm) each showed single symmetrical radioactive peaks at 16 cm from the origin following paper chromatography in System P-2 for 21 hours. Following acetylation, both acetates when chromatographed together in System P-5, gave single radioactive peaks with the same Rf value of 0.65. No ultraviolet absorption could be detected in either radioactive zone. An adjacently run marker of 208-dihydro-6$-08-F triacetate.had an Rf value of 0.15, No meaningful mass spectra were obtained from either acetate. Zone IV. The residue which contained 41,500 cpm, gave a single radioactive peak in HBV 1.5-2 following chromatography on 22 g Celite with System C-l. Repeated attempts to remove contaminating material by column chromatography and TLC were unsuccessful. Solvolyzable Fraction. Due to the small amount of radioactivity in relation to the large amount of contaminating pigments and the likelihood of multiple artifacts from the solvolytic procedure, no further analysis of this fraction was undertaken.
RESULTS AND DISCUSSION
Following the intravenous administration of l,2-3H-68-hydroxycertisol to an endocrinologically normal male, 34% of the injected dose was excreted in the urine during the first 24 hours (Table II).
Only an
additional 1.2% could be detected in the 48-hour collection and none in the 72-hour sample.
This excretion of radioactivity would appear to be
somewhat lower than the excretion encountered by Burstein et al. (3) who were
able to extract
collection.
59% of the injected dose from the 48-hour urine
These workers did not, however, measure the total amount of
radioactivity excreted.
During the present study, it was found possible
to extract 80% of the total urinary radioactivity by the use of tetrahydrofuran (Table II). remaining 20%.
It was not found possible to account for the
Hydrolysis of the urine with both S-glucuronidase and
sulfatase and subsequent ethyl acetate extraction did not yield any further amounts of radioactivity than were found in unhydrolyzed urine (Table II).
It was of interest to note that the amount of radioactivity
536
STEROIDS
13:4
extracted following hydrolysis was, in fact, less than that obtained without hydrolysis.
A possible explanation is the extra alkali wash to which
the hydrolyzed extract was subjected.
Although no significant amounts of
conjugated metabolites appeared to be present in the urine, extraction with tetrahydrofuran yielded 10% more radioactivity than ethyl acetate extsaction.
This can be explained on the assumption that some of the more
polar unidentified metabolites were not adequately extracted with ethyl acetate and a portion of the metabolites which were extracted were destroyed during alkali washing. Extraction of the urine at pH 1 with tetrahydrofuran, following previous extraction with the same solvent at pH 6, did not yield any further significant amounts of radioactivity after solvolysis with perchloric acid. This solvolytic procedure readily splits C-21 ester sulfates (7).
It can-
not be assumed, however, from this latter observation, that sulfated metabolites of 66-OH-F were not present.
Such metabolites would be extreme-
ly polar in nature and possibly not extracted by tetrahydrofuran at pH 1. In contrast to the in vivo metabolism of cortisol in the normal subject where approximately 70-80% of the administered dose is excreted in the urine in 24 hours, only 34% was excreted after the administration of 6@-OH-F.
It is possible that a sizeable portion of the remaining radio-
activity was excreted in the feces.
It is also of interest to note that
the major portion (57%) of the excreted radioactivity was found to be 6B-OH-F, whereas cortisol is primarily metabolized to tetrahydrocortisol and tetrahydrocortisone and followed by conjugation with glucuronic acid. The present study does demonstrate, however, that 68-OH-F, apart from its apparent lack of conjugated metabolites, does undergo several quantitatively minor chemical transformations.
These include oxidation of the
April 1969
STEROIDS
118-hydroxyl group and reduction of the C-20 carbonyl group to the 208configuration. Comparison of the specific activity of urinary 68-OH-F with those of the urinary 20B-dihydro-68-OH-F and 6#3-OH-E has indicated that 36% of urinary 2GB-dihydro-6%-08-F and 54% of urinary 68-OH-E were derived from circulating 6@-OH-F.
These results support the earlier suggestion that
two biochemical pathways are operative for the formation of 20B-dihydro-6SOM-F from cortisol:
the first involving 68-hydroxylation of 208-dihydro-
cortisol and the second, 20+reduction
of 68-OH-F.
t Present Address: Department of Obstetrics and Gynecology, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research Council of Great Britain. The authors are grateful to Mr. Stephen Jones, Department of Chemistry, Sheffield University, for determining the mass spectra. The secretarial assistance of Mrs. Selma J. Nagy, in the preparation of this manuscript, is also appreciated.
1.
Dixon, R. and Pennington, G.W., STEROIDS 12, 423 (1968).
2.
Dixon, R. and Pennington, G,W., J. ENDOCRINOL. 34, 281 (1966).
3.
Burstein, S., Kimball, H-L., Klaiber, E.L. and Gut, M., J. CLIN. ENDOCR. 27_, 491 (1967).
4.
Siiteri, P.K., STEROIDS 2, 687 (1963).
5.
Schneider, J.J. and Lewbart, M., TETRAHEDRON 20, 943 (1964).
6.
Frantz, A.G., Katz, F.H. and Jailer, J.W., J. CLIN. ENDOCR. 2, (1961).
1290
538
7.
STEROIDS
13:4
Burstein, S. and Lieberman, S., J. BIOL. CHEM. 233. 331 (1958).
GLOSSARY
The systematic names of steroids, identified in the text by trivial names or abbreviations, are: 6B-OH-F = 6B-hydroxycortisol = 68,11@,17cr,21-tetrahydroxypregn-4-ene-3,20dione. 2Oa(@)-dihydro-F = 20a(B)-dihydrocortisol = llB,17a,20a(B), 2l_tetrahydroxypregn-4-ene-3-one. 20a(@)-dihydro-68-OH-F = 68,118,17a,20a(8), 21-pentahydroxypregn-4-ene-3one. 6B-OH-E = 6@-hydroxycortisone = 68,17a,21-trihydroxypregn-4-ene-3,11,20trione.