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Metabolism of estriol-17α-3H in man
337
METABOLISM of ESTRIOL-17o~-3H in MAN Jack Fishman, Barnett Zumoff, Leon Hellman and T. F. Gallagher Institute for Steroid Research and the Division of Neoplastic Medicine, Montefiore Hospital and Medical Center, New York, N. V. 10467 Received December 30, 1967
ABSTRACT Estriol stereospecifically labelled with tritium at the 17oI position was prepared and administered together with 14C labelled estriol to three subjects including one cirrhotic. No significant tritium was present in the plasma or urine water and hence no oxidation at C-17 had occurred. Excretion of radioactivity was very rapid particularly in the cirrhotic subjects. Analysis of the enzyme and acid hydrolysates showed that they consisted of only three compounds. The majority of the excreted radioactivity consisted of estrio[ which did not lose any of the tritium at 17c~. The only other compounds were 16-ketoestradiol (0.8%) and 16-epiestriol (0.2%). Both metabolites retained the major part of the tritium at C-17ol and hence could not have arisen via 17-keto intermediates. The principal purpose of this study was to examine the possible participation of the C-17 hydroxyl group of estriol in oxidatlon-reduction reactions in vivo.
An earlier report by Levitz,
Spitzer and Twombly (1) showed that the 16~-hydroxyl group of this metabolite was oxidized to form 16-ketoestradiol and this product in turn was reduced to epiestriol. The amount of estriol so altered was less than 1% of the amount administered. The remainder was apparently unchanged estriol. These results were clarified by the kinetic studies of Sandberg and Slaunwhite (2) in normal and bilefistula patients. These authors showed that estriol was rapidly
338
ST ER O I D S
ellminatedl in urine chiefly as the glucosiduronatel and participated in an enterohepatlc circulation to a much smaller extent than estrone or estradiol. However they failed to find 16-ketoestradiol or epiestriol derived from estriol.
The present work confirmed the rapid elimination
of estriol, as well as its conversion to 16-ketoestradiol and epiestriol. In addition it was shown that these changes occurred with little loss of 3H from C-17 and that the estrlol excreted was virtually unaltered by oxidation-reduction at C-17. These results are in contrast with estradiol itself and with androgen metabolltes where the C-17 and C-3 hydroxyl groups have been shown to be extensively oxidized and to a considerable extent reduced back to its initial state during its biologic lifetime (3,4,5). EXPERIMENTAL Preparation of Estriol- 17~-3H. A solutian of 20 mg of l&z-hydroxyestrone in 5 ml of ethanol was treated with 5 mg of sodium borohydride-3H (5 mc/mg- New England Nuclear). After standing at room temperature for 1 hour, 15 ml of 5% sulfuric acid solution was added and the aqueous mixture was extracted 3 times with 25 ml of chloroform. The chloroform extract was dried and evaporated and the residue was submitted to preparative thin layer chromatography on silica gel containing zinc phosphor. The system used was ethyl acetate~ cyclohexane, ethanol 45:45:10. The estriol zone~ visualized under ultraviolet light1 was eluted with methanol and the solvent removed. The material was crystallized from dilute methanol and had a specific activity of 80.0 x 106 cpm/mg. A 0.608 mg portion was diluted with 52 mg of inert estrlol to give material with a calculated specific activity of 0.935 x 106 cpm/mg. Repeated crystallization of this material from dilute methanol yielded a product with constant specific activity of 0.95 x 106 cpm/mg. The combined mother liquors and crystals (52.6 rag), were dissolved in pyridine and treated with .033 ml of acetic anhydride (2.2 equivalents). After the usual workup the residue was separated by
11:3
March 1968
ST ER O I D S
preparative thin layer chromatography in ethyl acetate-cyclohexane 30:70. The area corresponding to estriol-3,16-diacetate was eluted and recrystallized from acetone-petroleum ether to give estriol-3,16-diacetate,specific activity of 0.945 x 106 cpm/mg corrected to the free compound. The crystalline estriol-3,16 diacetate (24 rag) was dissolved in 5 ml of freshly distilled acetone and treated with two drops of the Jones reagent~l After workup, the isolated 16~-hydroxyestrone diacetate was crystallized from acetone-petroleum ether to give material with a corrected specific activity of 28,000 cpm/mg. The specific activity did not change on subsequent crystallizations. Thus only 3% of the tritium was located elsewhere than at C-17o~. Subjects. Subject BO was a 50 year old man with cirrhosis of the liver of several years duration. Subjects PE and IO were women 72 and 74 years old with ca of the breast, and normal liver function. Preparation and Administration. The estriol-17cz-SH (80 pc/mg) was mixed with estriol-16-14C (20 pc/mg];~ the mixture was dissolved in propylene glycol to give a solution contain'~g 1.39 x 106 cpm of 14C and 5.06 x 106 x cpm of 3H per gram and a "~- ratio of 3.65. The solution was administered intravenously and urine collections were obtained at specified intervals for a total of four days. Plasma samples were obtained at intervals in the first 12 hours. Aliquots of each urine collection were counted for 14C and 3H content. Water o~tained by lyophilization of plasma and urine aliquots was counted for H content. A portion of the pooled urine collection was incubated with /3-glucuronldase (300 units/ml) pH 5.0 in acetate buffer at 38 ° for 5 days. The urine at pH 5 was extracted continuously with ether for 48 hours. The ether extract was washed with ice cold 9% sodium bicarbonate solution saturated with sodium chloride, with saturated sodium chloride solution and finally with water. The residue, the enzymic hydrolysate was counted for 14C and 3H content. The spent urine was adjusted to a 4.5% vol/vol sulfuric acid content and extracted continuously with ether for 72 hours. The ether extract was washed as previously and the residue, the acid hydrolysate, was counted for 14:Cand 3H content. A portion of each of the extracts of subject PE and of the enzyme hydrolysate of subject [O were separated by gradient el ution partition chromatography on acid washed Hyflo supercel column with 90% aqueous methanol as the stationery phase and trimethylpentane with a gradient of dichloroethane as the mobile phase according to
339
340
ST ER O ID S
Engel et al (9). Fractions of 10 ml were collected in an automatic fraction collector. The radioactive areas were located by counting aliquots with a windowless gas flow counter. Only three areas of radioactivity were detected in each instance (Fig 1). Fractions 110-121 corresponded to the ketol area consisting of 16-ketoestradiol and 16oz-hydroxyestrone. Fractions 128-140 contained ]6-epiestriol. Most of the radioactivity was contained in fractions 200-240 and represented estriol. Aliquots from each combined region were counted for 14C and 3H content. The 16-epiestriol region and an aliquot of the estriol region were diluted with the appropriate inert compounds. The ketol region was divided into two halves one of which was diluted with inert ]6ot-hydroxyestrone and the other with 16-ketoestradiol. All of the reverse isotope dilutions were purified further by preparative thin layer chromatography in ethylacetate. The eluted compounds were acetylated with acetic anhydride in pyridine and recrystallized from acetone-petroleum ether to constant specific activity and isotope ratio. The location of the tritium in the isolated estriol was established to be 17otonly, by a procedure identical with that used with the starting dose. RESULTS The rate of excretion of radioactivity measured from 14C was in agreement with previously published results (1,2) and only the total recovery is reported (Table 1). The recovery of 3H was equally good although this is not so precise a measurement when performed on raw urine. Urinary excretion of radioactivity was especially prompt with the cirrhotic patient in that 72% of the 14C administered (or 80% of the total recovered in urine) was eliminated during the first 8 hours of the study. No measurable radioactivity was found in the water obtained by lyophilization of blood and urine specimens obtained throughout the study. This together with essentially comparable recovery of each isotope in the excretion products established that little biochemical oxidation of C-17 had occurred.
11:3
341
C"4
0
0
L-
.,4,
0 CO
0 0 O r.)
0 tO)
-<
¢I)
Z 0
J
IJ.
0 0',
L..}
,'.~-
C
~
C ,,@
0
0
342
ST ER O ID S
11:3
TABLE I Subject
Urine*
Enzyme Hydrolysis
Acid Hydrolysis
BO
89.0
43.0
9.0
PE
84.0
64.0
4.5
tO
90.0
73.0
6.8
*Excretion during four days. All values are percentages of 14C portion of dose.
TABLE II
3H/14C
% A3H
Yield % Dose
67.0
Dose
3.7
Estriol *
3.8
+4
16-Epiestriol*
3.2
- 10
0.20
16-Ketoestradiol *
2.8
-25
0.80
2.8
-25
0.10
16-Ketoestradiol
#
*Obtained from Enzyme Hydrolysate. #Obtained from Acid Hydrolysate.
March 1968
STEROIDS
In the partition chromatogram of the two urine extracts that were examined for metabolites only three areas of radioactivity were detected (Fig 1). The principal amount was estriol as shown by carrier addition and recrystallization to constant specific activity. Since all the 14C and 3H remained with the carrier estdol no other product was present in this area of the chromatogram. The location of the tritium was found to be unchanged from that of the starting material. The other two peaks of radioactivity were well separated from each other and from estriol. The polar area, from its position in the chromatogram was epiestriol.
Carrier addition followed by recrystallization established
that over 90% of the 14C radioactivity and very nearly the same percent of 3H was in fact in epiestriol. The less polar area contained the ring D ketols. Separate portions of this material were mixed with inactive 16~hydroxyestrone and 16-ketoestradiol. Recrystallization of these products demonstrated that virtually all the 14C-radioactivity and most of the 3H was associated with 16-ketoestrad|ol. The 16~-hydroxyestrone was virtually devoid of 14C or 3H. (Table ]I). DISCUSSION The conclusions that can be drawn from these studies are quite clear. Administered estriol, apart from coniugation, is metabolically unaltered by any oxidative reaction at C-17. A very small fraction is converted to 16-ketoestradiol, in agreement with the results of Levitz and Twombly, (1) and an even smaller fraction is converted to epiestriol. The almost complete retention of isotope
343
344
ST ER O ID S
virtually excludes a 17-ketone as intermediate in these transformations. This lack of participation in an in vivo oxldatlon-reduction system distinguishes estrlol from many other hydroxyl~ted steroids and their metabolites. The fact that the 3H:14C ratio was slightly altered in the epiestriol compared with the administered metabolite may be due to some incorporation of 3H at C-16 during the preparation of the labeled steroid. The reaction leading to 17~-3H-estriol, while highly stereoselective, is only 97% stereospeclfic, it may be that the remaining isotope was at C-16 and this loss together with the errors inherent in double isotope counting procedures was responsible for the apparent small loss in the transformation. The somewhat greater loss in the transition to 16-ketoestradiol may result in part from the same factors as well as an additional possibility. If at any time during the collection or storage period in these studies, the urine became alkaline it is very probable that some enolization of the ]6-ketone occurred. Even though minor, this could account for the observed difference between the isotope ratios of estriol and 16-ketoestradiol. It is significant that acid catalyzed enollzatlon did not cause loss of 3H since the 16-ketoestradlol from the enzymic hydroJysate and from the cold acid hydrolysate had precisely the same isotope ratio. Thus it seems very probable that the isolation procedures maintained an isotoplcally stable product. With these minor uncertainties, it is important to emphasize the major retention of the isotope at C-17 in a potentially labile ketol metabolite.
11:3
March 1968
STEROIDS
It is clear from prior studies that the major" pathway in the formation of epiestriol is hydroxylation of estrone at the ]6/3-orlentatlon (7). The present studies affirm this in the sense that formation from estriol~ although demonstrated~ is far too small to account for the endogenous production from estradiol alone and neglecting other potential precursors. It can be estimated from available data that at a maximum only 2% of the excreted epiestrio[ is derived from estriol with the remainder coming by the main route involving 17-keto intermediates. It seems probable that a similar statement could be made about 16-ketoestradiol but estimations of the daily production of this metabolite are fragmentary and probably inherently unreliable because of the possibility of rearrangement of 16/%hydroxyestrone to this compound. The rapid excretion of estriol in the cirrhotic patient was expected because the metabolltes of estradiol appear more promptly in the urine of these patients {8). The rapid initial excretion of radioactivity after estriol injection is also consistent with Sandberg and Slaunwhite's (2) interpretation of the lesser enterohepatic circulation of this compound. This enterohepatic circulation thus is probably still smaller in the cirrhotic subject. The lack of oxidation of estriol at C-17 in the cirrhotic patient (as evident from the lack of radioactivity in plasma or urine water) is of particular significance since it has been demonstrated (8) that in cirrhosis the amounts of 16c~-hydroxyestrone are substantially increased. From the present results it is clear that this increase is not due to oxidation of estriol.
345
ST ER O ID S
346
ACKNOWLEDGMENTS This work was supported by a grant from the American Cancer Society and by Grants CA-07304 from the National Cancer Institute and FR-53 from the General Clinical Research Centers Branch, National Institutes of Health, U.S.P.H.S. The authors wish to thank Mrs. R. Lehman, Mrs. O. Gurny and Mrs. Gertrude Gilman for their most able technical assistance.
REFERENCES .
Levitz, M., Spitzer, J. R., and Twombly, G. H., J. BIOL. CHEM. 231, 787 (1958).
.
Sandberg, A. A., and Slaunwhlte, W. R. Jr., J. CLIN. INVEST.
44, 644 (1965). .
Fishman, J., Bradlow, H. L., and Gallagher, T. F., J. BIOL. CHEM. 235, 3104 (1960).
.
Bradlow, H. L., Fukushlma, D. K., Zumoff, B., Cassouto, J., Hellman, U, and Gallagher, T. F., J. CLIN. ENDOCR. 27, 1203 (1967).
.
Fukushima, D. K., Bradlow, H. U, Yamauchi, T., Zumoff, B., Hellman, L., and Gallagher, T. F., IBID 27, 1208 (1967).
.
Bowden, K., Heilbron, I. M., Jones, E. R. H., and Weedon, B.C.L., J. CHEM. SOC. 39 (1946).
.
Fishman, J., Hellman, L., Zumoff, B., and Cassouto, J., BIOCHEMISTRY 5, 1784 (1966).
.
.
Zumoff, B., Fishman, J., Hellman, L., and Gallagher, T. F. J. CLIN. INVEST. IN PRESS (Jan. 1968). Engel, U L., Cameron, C. B., Stofyn, A., Alexander, J. A., Klein, O., and Troflmow, N. A., ANAL. BIOCHEM. 2, 114 (1961).
11:3
METABOLISM of ESTRIOL-17o~-3H in MAN Jack Fishman, Barnett Zumoff, Leon Hellman and T. F. Gallagher Institute for Steroid Research and the Division of Neoplastic Medicine, Montefiore Hospital and Medical Center, New York, N. V. 10467 Received December 30, 1967
ABSTRACT Estriol stereospecifically labelled with tritium at the 17oI position was prepared and administered together with 14C labelled estriol to three subjects including one cirrhotic. No significant tritium was present in the plasma or urine water and hence no oxidation at C-17 had occurred. Excretion of radioactivity was very rapid particularly in the cirrhotic subjects. Analysis of the enzyme and acid hydrolysates showed that they consisted of only three compounds. The majority of the excreted radioactivity consisted of estrio[ which did not lose any of the tritium at 17c~. The only other compounds were 16-ketoestradiol (0.8%) and 16-epiestriol (0.2%). Both metabolites retained the major part of the tritium at C-17ol and hence could not have arisen via 17-keto intermediates. The principal purpose of this study was to examine the possible participation of the C-17 hydroxyl group of estriol in oxidatlon-reduction reactions in vivo.
An earlier report by Levitz,
Spitzer and Twombly (1) showed that the 16~-hydroxyl group of this metabolite was oxidized to form 16-ketoestradiol and this product in turn was reduced to epiestriol. The amount of estriol so altered was less than 1% of the amount administered. The remainder was apparently unchanged estriol. These results were clarified by the kinetic studies of Sandberg and Slaunwhite (2) in normal and bilefistula patients. These authors showed that estriol was rapidly
338
ST ER O I D S
ellminatedl in urine chiefly as the glucosiduronatel and participated in an enterohepatlc circulation to a much smaller extent than estrone or estradiol. However they failed to find 16-ketoestradiol or epiestriol derived from estriol.
The present work confirmed the rapid elimination
of estriol, as well as its conversion to 16-ketoestradiol and epiestriol. In addition it was shown that these changes occurred with little loss of 3H from C-17 and that the estrlol excreted was virtually unaltered by oxidation-reduction at C-17. These results are in contrast with estradiol itself and with androgen metabolltes where the C-17 and C-3 hydroxyl groups have been shown to be extensively oxidized and to a considerable extent reduced back to its initial state during its biologic lifetime (3,4,5). EXPERIMENTAL Preparation of Estriol- 17~-3H. A solutian of 20 mg of l&z-hydroxyestrone in 5 ml of ethanol was treated with 5 mg of sodium borohydride-3H (5 mc/mg- New England Nuclear). After standing at room temperature for 1 hour, 15 ml of 5% sulfuric acid solution was added and the aqueous mixture was extracted 3 times with 25 ml of chloroform. The chloroform extract was dried and evaporated and the residue was submitted to preparative thin layer chromatography on silica gel containing zinc phosphor. The system used was ethyl acetate~ cyclohexane, ethanol 45:45:10. The estriol zone~ visualized under ultraviolet light1 was eluted with methanol and the solvent removed. The material was crystallized from dilute methanol and had a specific activity of 80.0 x 106 cpm/mg. A 0.608 mg portion was diluted with 52 mg of inert estrlol to give material with a calculated specific activity of 0.935 x 106 cpm/mg. Repeated crystallization of this material from dilute methanol yielded a product with constant specific activity of 0.95 x 106 cpm/mg. The combined mother liquors and crystals (52.6 rag), were dissolved in pyridine and treated with .033 ml of acetic anhydride (2.2 equivalents). After the usual workup the residue was separated by
11:3
March 1968
ST ER O I D S
preparative thin layer chromatography in ethyl acetate-cyclohexane 30:70. The area corresponding to estriol-3,16-diacetate was eluted and recrystallized from acetone-petroleum ether to give estriol-3,16-diacetate,specific activity of 0.945 x 106 cpm/mg corrected to the free compound. The crystalline estriol-3,16 diacetate (24 rag) was dissolved in 5 ml of freshly distilled acetone and treated with two drops of the Jones reagent~l After workup, the isolated 16~-hydroxyestrone diacetate was crystallized from acetone-petroleum ether to give material with a corrected specific activity of 28,000 cpm/mg. The specific activity did not change on subsequent crystallizations. Thus only 3% of the tritium was located elsewhere than at C-17o~. Subjects. Subject BO was a 50 year old man with cirrhosis of the liver of several years duration. Subjects PE and IO were women 72 and 74 years old with ca of the breast, and normal liver function. Preparation and Administration. The estriol-17cz-SH (80 pc/mg) was mixed with estriol-16-14C (20 pc/mg];~ the mixture was dissolved in propylene glycol to give a solution contain'~g 1.39 x 106 cpm of 14C and 5.06 x 106 x cpm of 3H per gram and a "~- ratio of 3.65. The solution was administered intravenously and urine collections were obtained at specified intervals for a total of four days. Plasma samples were obtained at intervals in the first 12 hours. Aliquots of each urine collection were counted for 14C and 3H content. Water o~tained by lyophilization of plasma and urine aliquots was counted for H content. A portion of the pooled urine collection was incubated with /3-glucuronldase (300 units/ml) pH 5.0 in acetate buffer at 38 ° for 5 days. The urine at pH 5 was extracted continuously with ether for 48 hours. The ether extract was washed with ice cold 9% sodium bicarbonate solution saturated with sodium chloride, with saturated sodium chloride solution and finally with water. The residue, the enzymic hydrolysate was counted for 14C and 3H content. The spent urine was adjusted to a 4.5% vol/vol sulfuric acid content and extracted continuously with ether for 72 hours. The ether extract was washed as previously and the residue, the acid hydrolysate, was counted for 14:Cand 3H content. A portion of each of the extracts of subject PE and of the enzyme hydrolysate of subject [O were separated by gradient el ution partition chromatography on acid washed Hyflo supercel column with 90% aqueous methanol as the stationery phase and trimethylpentane with a gradient of dichloroethane as the mobile phase according to
339
340
ST ER O ID S
Engel et al (9). Fractions of 10 ml were collected in an automatic fraction collector. The radioactive areas were located by counting aliquots with a windowless gas flow counter. Only three areas of radioactivity were detected in each instance (Fig 1). Fractions 110-121 corresponded to the ketol area consisting of 16-ketoestradiol and 16oz-hydroxyestrone. Fractions 128-140 contained ]6-epiestriol. Most of the radioactivity was contained in fractions 200-240 and represented estriol. Aliquots from each combined region were counted for 14C and 3H content. The 16-epiestriol region and an aliquot of the estriol region were diluted with the appropriate inert compounds. The ketol region was divided into two halves one of which was diluted with inert ]6ot-hydroxyestrone and the other with 16-ketoestradiol. All of the reverse isotope dilutions were purified further by preparative thin layer chromatography in ethylacetate. The eluted compounds were acetylated with acetic anhydride in pyridine and recrystallized from acetone-petroleum ether to constant specific activity and isotope ratio. The location of the tritium in the isolated estriol was established to be 17otonly, by a procedure identical with that used with the starting dose. RESULTS The rate of excretion of radioactivity measured from 14C was in agreement with previously published results (1,2) and only the total recovery is reported (Table 1). The recovery of 3H was equally good although this is not so precise a measurement when performed on raw urine. Urinary excretion of radioactivity was especially prompt with the cirrhotic patient in that 72% of the 14C administered (or 80% of the total recovered in urine) was eliminated during the first 8 hours of the study. No measurable radioactivity was found in the water obtained by lyophilization of blood and urine specimens obtained throughout the study. This together with essentially comparable recovery of each isotope in the excretion products established that little biochemical oxidation of C-17 had occurred.
11:3
341
C"4
0
0
L-
.,4,
0 CO
0 0 O r.)
0 tO)
-<
¢I)
Z 0
J
IJ.
0 0',
L..}
,'.~-
C
~
C ,,@
0
0
342
ST ER O ID S
11:3
TABLE I Subject
Urine*
Enzyme Hydrolysis
Acid Hydrolysis
BO
89.0
43.0
9.0
PE
84.0
64.0
4.5
tO
90.0
73.0
6.8
*Excretion during four days. All values are percentages of 14C portion of dose.
TABLE II
3H/14C
% A3H
Yield % Dose
67.0
Dose
3.7
Estriol *
3.8
+4
16-Epiestriol*
3.2
- 10
0.20
16-Ketoestradiol *
2.8
-25
0.80
2.8
-25
0.10
16-Ketoestradiol
#
*Obtained from Enzyme Hydrolysate. #Obtained from Acid Hydrolysate.
March 1968
STEROIDS
In the partition chromatogram of the two urine extracts that were examined for metabolites only three areas of radioactivity were detected (Fig 1). The principal amount was estriol as shown by carrier addition and recrystallization to constant specific activity. Since all the 14C and 3H remained with the carrier estdol no other product was present in this area of the chromatogram. The location of the tritium was found to be unchanged from that of the starting material. The other two peaks of radioactivity were well separated from each other and from estriol. The polar area, from its position in the chromatogram was epiestriol.
Carrier addition followed by recrystallization established
that over 90% of the 14C radioactivity and very nearly the same percent of 3H was in fact in epiestriol. The less polar area contained the ring D ketols. Separate portions of this material were mixed with inactive 16~hydroxyestrone and 16-ketoestradiol. Recrystallization of these products demonstrated that virtually all the 14C-radioactivity and most of the 3H was associated with 16-ketoestrad|ol. The 16~-hydroxyestrone was virtually devoid of 14C or 3H. (Table ]I). DISCUSSION The conclusions that can be drawn from these studies are quite clear. Administered estriol, apart from coniugation, is metabolically unaltered by any oxidative reaction at C-17. A very small fraction is converted to 16-ketoestradiol, in agreement with the results of Levitz and Twombly, (1) and an even smaller fraction is converted to epiestriol. The almost complete retention of isotope
343
344
ST ER O ID S
virtually excludes a 17-ketone as intermediate in these transformations. This lack of participation in an in vivo oxldatlon-reduction system distinguishes estrlol from many other hydroxyl~ted steroids and their metabolites. The fact that the 3H:14C ratio was slightly altered in the epiestriol compared with the administered metabolite may be due to some incorporation of 3H at C-16 during the preparation of the labeled steroid. The reaction leading to 17~-3H-estriol, while highly stereoselective, is only 97% stereospeclfic, it may be that the remaining isotope was at C-16 and this loss together with the errors inherent in double isotope counting procedures was responsible for the apparent small loss in the transformation. The somewhat greater loss in the transition to 16-ketoestradiol may result in part from the same factors as well as an additional possibility. If at any time during the collection or storage period in these studies, the urine became alkaline it is very probable that some enolization of the ]6-ketone occurred. Even though minor, this could account for the observed difference between the isotope ratios of estriol and 16-ketoestradiol. It is significant that acid catalyzed enollzatlon did not cause loss of 3H since the 16-ketoestradlol from the enzymic hydroJysate and from the cold acid hydrolysate had precisely the same isotope ratio. Thus it seems very probable that the isolation procedures maintained an isotoplcally stable product. With these minor uncertainties, it is important to emphasize the major retention of the isotope at C-17 in a potentially labile ketol metabolite.
11:3
March 1968
STEROIDS
It is clear from prior studies that the major" pathway in the formation of epiestriol is hydroxylation of estrone at the ]6/3-orlentatlon (7). The present studies affirm this in the sense that formation from estriol~ although demonstrated~ is far too small to account for the endogenous production from estradiol alone and neglecting other potential precursors. It can be estimated from available data that at a maximum only 2% of the excreted epiestrio[ is derived from estriol with the remainder coming by the main route involving 17-keto intermediates. It seems probable that a similar statement could be made about 16-ketoestradiol but estimations of the daily production of this metabolite are fragmentary and probably inherently unreliable because of the possibility of rearrangement of 16/%hydroxyestrone to this compound. The rapid excretion of estriol in the cirrhotic patient was expected because the metabolltes of estradiol appear more promptly in the urine of these patients {8). The rapid initial excretion of radioactivity after estriol injection is also consistent with Sandberg and Slaunwhite's (2) interpretation of the lesser enterohepatic circulation of this compound. This enterohepatic circulation thus is probably still smaller in the cirrhotic subject. The lack of oxidation of estriol at C-17 in the cirrhotic patient (as evident from the lack of radioactivity in plasma or urine water) is of particular significance since it has been demonstrated (8) that in cirrhosis the amounts of 16c~-hydroxyestrone are substantially increased. From the present results it is clear that this increase is not due to oxidation of estriol.
345
ST ER O ID S
346
ACKNOWLEDGMENTS This work was supported by a grant from the American Cancer Society and by Grants CA-07304 from the National Cancer Institute and FR-53 from the General Clinical Research Centers Branch, National Institutes of Health, U.S.P.H.S. The authors wish to thank Mrs. R. Lehman, Mrs. O. Gurny and Mrs. Gertrude Gilman for their most able technical assistance.
REFERENCES .
Levitz, M., Spitzer, J. R., and Twombly, G. H., J. BIOL. CHEM. 231, 787 (1958).
.
Sandberg, A. A., and Slaunwhlte, W. R. Jr., J. CLIN. INVEST.
44, 644 (1965). .
Fishman, J., Bradlow, H. L., and Gallagher, T. F., J. BIOL. CHEM. 235, 3104 (1960).
.
Bradlow, H. L., Fukushlma, D. K., Zumoff, B., Cassouto, J., Hellman, U, and Gallagher, T. F., J. CLIN. ENDOCR. 27, 1203 (1967).
.
Fukushima, D. K., Bradlow, H. U, Yamauchi, T., Zumoff, B., Hellman, L., and Gallagher, T. F., IBID 27, 1208 (1967).
.
Bowden, K., Heilbron, I. M., Jones, E. R. H., and Weedon, B.C.L., J. CHEM. SOC. 39 (1946).
.
Fishman, J., Hellman, L., Zumoff, B., and Cassouto, J., BIOCHEMISTRY 5, 1784 (1966).
.
.
Zumoff, B., Fishman, J., Hellman, L., and Gallagher, T. F. J. CLIN. INVEST. IN PRESS (Jan. 1968). Engel, U L., Cameron, C. B., Stofyn, A., Alexander, J. A., Klein, O., and Troflmow, N. A., ANAL. BIOCHEM. 2, 114 (1961).
11:3