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Metabolism of 3H-digitoxigenin by rat liver, adrenal, and ovary homogenates
87
METABOLISM OF 3H-DIGITOXIGENIN BY RAT LIVER, ADRENAL, AND OVARY HOMOGENATES R.E. Talcott, W.H. Bulger, and S.J. Stohs Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Nebraska, Lincoln, Nebraska 68508 Received: ABSTRACT The metabolism of 3H-digitoxigenin was studied in rat liver, adrenal, and ovary homogenates under identical conditions. The major metabolite formed by liver and ovarian preparations was 3epidigitoxigenin. Male liver homogenates showed higher epimerizing activity than female liver or ovary homogenates. In the adrenal preparations, the major metabolite formed was 3-digitoxigenone, and no sex difference was observed in its rate of formation. Adrenal and liver homogenates produced small amounts of digitoxigenin polar metabolites. The polar metabolites formed by the adrenal preparations were tentatively identified as fhydroxydigitoxigenin and 168-hydroxydigitoxigenin.
INTRODUCTION The cardioactive steroid digitoxigenin 58,148-card-20(22)-enolide) lide type are biotransformed
(38,14-dihydroxy-
and other steroids of the cardenoto the pharmacologically
inactive
3a-hydroxy epimers by the 3-hydroxysteroid dehydrogenases found in rat liver (1,2,3). steps:
The epimerization takes place fn two
First, the substrate, having the 3-hydroxyl group in the
8 configuration, is oxidized to the corresponding 3-keto compound by 3B-hydroxysteroid dehydrogenase
(E.C..l.l.l.51).
The
3-keto compound is then reduced to the 3a-hydroxyl compound by 3ahydroxysteroid dehydrogenase
(E.C. 1.1.1.50).
The pyridine nuc-
leotide coenzymes apparently serve as hydrogen carriers in these steps (1,3).
STEROIDS
88
21:l
In the synthesis of steroid hormones, the conversion of pregnenolone to progesterone involves the conversion of a B-hydroxyl group to a carbonyl group at position 3 and a subsequent rearrangement of a double bond from A5 to A4. This conversion has been demonstrated -in vitro with rat ovarian tissue (4) and adrenal tissue (5). Furthermore, the conversfon of 38-hydroxypregnanesto their 3a-hydroxy epimers has been recently demonstrated in rat ovarian cytosol (6). The oxidoreductases that catalyze these conversions may al.80metabolize digitoxfgenin, To examine this possibility, we have investigated the metabolism of digitoxfgenin by rat liver, adrenal, and ovary homogenates under fdentfcal incubation conditions.
A generous supply of 3H-digitoxigeninwas provfded by Gesellschaft fur Kernforschung, M.B.H., Germany. The speciffc activity was 655 niC/mg. Thfs compound was purified by preparative thin layer chromatography (TLC) to a radfochemicaf purity of greater than 98 per cent by methods prevfousllydescribed (2). Male and fernaLerats derived from the Holtzmann strain were obtained from ,Sasco,Enc., Omaha, Nebraska and were maintained on Purina Laboratory Chow and tap water, ad libitum. A total of 35 male and 35 female rats, 255-225 gm, were used Pm these experiments. Animals were fasted overnight prior to sacrifice. All animals were sacrificed between 7:30 and 8:00 A.M. by cervical.dislocation and exsanguination. Tissue was quickly excised and immediately placed in ice-cold 0.25 M sucrose 9 TMN (5.55 M Tris chloride, pH 7.4, 0.005 M MgCP2$ and 0.010 M NaCP) buffer. Tissue pooled from 15 rats was used to prepare Ifver, ovary, and adrenal homogenates. Xn each case, tissue (1 gm/lZ ml) was homogenized in 0.25 M sucrose + TMN solution using a Potter-Elvehjem homogenizer, and 2 ml of the resulting homogenates were added to each of three 2.5ml Erlenmeyer flasks tontaining the incubation mixture. Each flask contained 1.25 ml 0.25 M sucrose + TMN buffer in which were dissolved 3.5 mg NADP+, 14 mg glucose &-phosphate, .5 units glucose &phosphate dehydrogenase, 7.3 mg nicotinamide, and 15 mg bovine serum albumfn. One mg of carrier digitoxigenin and 0.5 PC 3H-d~g~toxigeninwere added to the fncubatiou mixture Pn 0.2 ml 95% ethanol. AlI.flasks were Incubated for 30 min on an
Jan. 1973
STEROIDS
89
Eberbach water bath shaker at 37OC, gassing the flasks with 95% O2-5% C02. The incubations were stopped and 3H-digitoxigenin and metabolites were extracted with methylene dichloride and assayed by TLC and liquid scintillation counting as previously described (2). For each tissue, the per cent conversion to the digitoxigenin major metabolite was calculated, and results were expressed as nmoles product formed/min/lOO mg tissue. Student's t-test was used to compare rates obtained with female liver and adrenals to those obtained with male liver and adrenals (see Table 1). The experiment was repeated once and the results pooled, accounting for the 6 determinations/tissue indicated in Table 1. A small amount of the radioactivity in the male and female adrenal and liver homogenate extracts (0.5-2%) was localized in a low Rf band, indicating that small amounts of digitoxigenin polar metabolites had been formed during the incubations. The adrenal extracts were pooLed and concentrated to dryness. The residue was redissolved in approximately 0.5 ml of methylene dichloride-methanol (1:1), and spotted on a preparative silica gel H plate (0.5 mm thick) which was developed once in chloroform-isopropanol (9:l). The polar metabolite band, Pocalized by the method of Bhavnani (7), was removed from the plate, placed in a glass-stoppered 250 ml Erlenmeyer flask containing 50 ml methylene dichloride-methanol (1:1), and extracted 2 hr on a shaker. This procedure was found to remove all of the radioactivity from the gel. The silica gel was removed by filtration. The filtrate was concentrated to dryness, in vacua, the residue was redissolved in 2 ml of methylene dichloride-methanol (l:l), and this solution was tested by TLC, developing a silica gel II plate 4 times in chloroform-isopropanol (9:l). Upon spraying the plate with 20% ll2SO4 and heating it at llO" for 5 min, two spots of different color and about equal intensity resolved. The colors and Rf values obtained suggested to us that these polar metabolites may be 5-hydroxydigitoxigenin (periplogenin)+c and 168-hydroxydigitoxigenin (gitoxigenin).** Therefore, the extract from the purification described above was concentrated to dryness and the preparative procedure was repeated, using authentic 5-hydroxydigitoxigenin and 16+hydroxydigftoxigenin as marker steroids and removing the areas on the preparative plate (developed 4 times in chloroform-isopropanol (9:l)) corresponding to them. The two metabolites isolated (Pletabolite I and MetaboPite II> were co-chromatographed with standards on a silica gel H plate developed 4 times in the above-mentioned solvent system, Co-chromatography was also performed in a second system using silica gel I plates developed 4 times in cyclohexane-acetone-acetic acid (65:33:2): Spots were visualized with either 20% H2SO4 or anisaldehyde sp&y reagent (0.5 ml anisaldehyde, 1.0 ml cont. H2SO4, and 50 ml glacial acetic acid). Using the latter solvent system and the two spray reagents, we were previously able to distinguish between 13 digitoxigenin derivatives (8). The results of the chromatographic analyses are presented in Table 2. ,
STEROIDS
90
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RESULTS The major metabolites of digitoxigenin and their rates of formation obtained with homogenates of various tissues are given in Table 1. TABLE 1 Metabolism of 'H-Digitoxlgenin by Tissue Homogenates from Male and Female Rats
Tissue*
3-Digitoxigenone formation (nmoles/min/lOOmg tissue)
3-Ep~d~g~tox~gen~n formation (nmoles/mfn/l00mg tissue)
Adrenals (male)
6.34 + 0,55
0.0
Adrenals (female)
6.99 2 0.87"
0.0
Liver
0.0
(male)
14.X.02 1.74
Liver (female)
0.0
4.40 " 0.88
Ovaries
0.0
4.20 -4_ 0.83
*n = 6 determinations 1n.s. with respect to male adrenal homogenates 2p 0.001 with respect to male liver homogenates
In liver homogenates obtained from rats of either Bex, 3-epidigitoxigenfn* was the major product. The results indicate that epimerizatfon proceeded more rapidly fn male liver homog in female liver homogenates, Epimerizatfon was also obs ovary homogenates, at a rate simflar to that observed wfth female liver homogenates. Adrenal homogenates obtained from either
91
STEROIDS
Jan. 1973
TABLE 2 Co-chromatographyof 3H-Digitoxigenin Polar Metabolites Formed by Adrenal Homogenates Obtained from Male and Female Rats
Rf
Compound(s)
Color
System 11 System XI2 H2SO4 Aniealdehyde
Metabolfte L
0.48
0.40
green
blue
5-hydroxydigitoxigenin
0.48
0.40
green
blue
5+ydroxydigitoxEgenin
0.48
0.40
green
blue
Metabolite XX
0.54
0.38
purple
green
16&-hydroxydigitoxigenin
0.54
0.38
purple
green
Hetabolite EL + 16S-hydroxydlgitoxigenin
0.54
0.38
purple
green
Metabolite I f
'silica gel H plates developed 4 times Pn chloroform-isopropanol 2(9:1) silica gel H plates developed 4 times in cyclohexane-acetone-acetic acid (65:33:2)
male or female rats primarily formed 3-digitoxigenone**with the oxidation occuring at similar rates in each sex. In addition to these metabolites, the adrenal and liver preparations obtained from rats of either sex formed small.amounts (0.5-Z% conversion) of polar digitoxigenin derivatives. The two polar metabolites formed by the adrenal homogenates were isolated
21:l
STEROIDS by preparative TLC (see experimental section) and examined by TLC in two solvent systems using two color-producing spray reagents. The Rf values and colors obtained with Metabolites I and II were identical with those obtained with authentic 5-hydroxydigitoxigenin and 16B-hydroxydigitoxigeninrespectively (Table 2).
The pro-
duction of these two metabolites, as well as several other previously unreported derivatives of digitoxigenin has been recently observed in rat liver homogenates and microsomes (Talcott and Stohs, in preparation). Furthermore, rabbit liver homogenates also produce large amounts of hydroxylated products of digitoxigenin, one of which is 5-hydroxydigitoxigenin(Bulger and Stohs, in preparation). DISCUSSION The rate-limiting step in the 3-epimerizationof cardenolides and other 3$-hydroxysteroidsby rat liver homogenates is the oxidation of the substrate to the 3-keto intermediate by 3S+ydroxysteroid dehydrogenase. This intermediate is almost immediately reduced to the epimer by 3a-hydroxysteroiddehydrogenase (1). Hence, with digitoxigenin as the substrate, appreciable amounts of 3-digitoxigenone do not accumulate in liver homogenates incubated at physiological pH (1,Z). In this study, we observed production of 3-epidigitoxigenin by ovary homogenates (Table 1) as well as by liver homogenates with no accumulation of the 3-keto intermediate. These results suggest that the epimerizing system recently described in rat ovaries (6) is similar to the system present in rat liver. In contrast, epimerization did not occur in adrenal homogenates and the major metabolite was 3-digitoxigenone. If a 3a-hydroxy-steroid
Jan. 1973
STEROIDS
dehydrogenase is present in rat adrenal homogenates, it apparently cannot reduce 3-digitoxigenoneunder our conditions. Repke and Samuels have previously observed a sex difference in 3B-hydroxysteroidepimerization and have attributed it to the "absent or exceptionally low activity" of the 3Phydroxysteroid dehydrogenase in female rat liver (1). In this regard, it is interesting that no sex difference in 3Phydroxysteroid dehydrogenase activity was noted in adrenal homogenates (see Table 1), suggesting that the liver and adrenal 3B-hydroxysteroiddehydrogenases are regulated differently. These results might be anticipated if digitoxigenin is oxidized by the same adrenal dehydrogenase that catalyzes the 3-dehydrogenationof pregnenolone piror to its conversion to progesterone. Since this step fs critical to the syntehsis of corticosteroids in both sexes, it is unlikely that 3B-hydroxysteroiddehydrogenase activity would be markedly different in male and female adrenal homogenates. The two polar derivatives of digitoxigenin produced by adrenal homogenates have been tentatively identified as 5-hydroxydigitoxigenin and 16$-hydroxydigitoxigeninby co-chromatographyof the metabolites with standards (Table 2). Rat liver homogenates also generate polar metabolites of digitoxigenin and the conversion can be greatly enhanced by prior treatment of the animals with phenobarbital (2). Preliminary evidence indicates that these polar metabolites include 5-hydroxydigitoxigeninand 16g-hydroxydigitoxPgenin plus several other derivatives, as yet unidentified, Work on positive identificationof the polar metabolites of digitoxigenin produced by rat and rabbit liver homogenates is currently in progress.
93
STEROIDS
94
An earlier finding of Repke (9) may have bearing on the pharmacodynamic significance of the results presented here. He found that 3 minutes after i.v. injection of the cardiac glycoside digitoxin, the concentration of drug found in the adrenal glands was 7-8 times that found in the liver (9). The fate of digitoxin in the adrenals is not known, but if significant amounts of it are hydroxylated on the steroid nucleus, or if the glycoside bonds can be cleaved, exposing the 38-hydroxyl group to oxidation, then the reactions which occur in adrenal homogenates with digftoxigenin as substrate may play a role in the metabolism of digitoxin.
ACKNOWLEDGEMENTS This investigation was supported in part by a National Institutes of Health Predoctoral Fellowship, #5F01 GM47428, from the National Institute of General Medical Sciences, a grant from the Nebraska Heart Association, and an Edwin Leigh Newcomb Fellowship from the American Foundation for Pharmaceutical Education.
REFERENCES 1.
Repke, K;, and Samuels, L.T., BIOCHEMISTRY 3, 689 (1964).
2.
Stohs, S.J., Reinke, L.A., and El-Olemy, M.M., BIOCHEM. PHARMACOL. 20, 473 (1971).
3.
Talcott, R.E., Stohs, S.J., and El-Olemy, M.M., BIOCHEM. PHARMACOL. 21, 2001 (1972).
4.
Huang, W.Y., and Pearlman, W.H., J. BIOL. CHEM. 237, I060 (1962).
5.
Rubin, B.L., and Dorfman, R.I., ENDOCRINOLOGY 61, 601 (1957).
6.
Nimrod, A., and Lindner, H.R., BIOCHEM. BIOPHYS. ACTA 261, 291 (1972).
7.
Bhavnani, B.R., J. CHROMATOG. 65, 454 (1972).
8.
Bulger, W.H., Talcott, R.E., and Stohs, S.J., J. CHROMATOG. 70, 187 (1972).
9.
Repke, K., ARCH. EXP. PATHOL. PHARMAKOL. 241, 165 (1961).
21:l
METABOLISM OF 3H-DIGITOXIGENIN BY RAT LIVER, ADRENAL, AND OVARY HOMOGENATES R.E. Talcott, W.H. Bulger, and S.J. Stohs Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Nebraska, Lincoln, Nebraska 68508 Received: ABSTRACT The metabolism of 3H-digitoxigenin was studied in rat liver, adrenal, and ovary homogenates under identical conditions. The major metabolite formed by liver and ovarian preparations was 3epidigitoxigenin. Male liver homogenates showed higher epimerizing activity than female liver or ovary homogenates. In the adrenal preparations, the major metabolite formed was 3-digitoxigenone, and no sex difference was observed in its rate of formation. Adrenal and liver homogenates produced small amounts of digitoxigenin polar metabolites. The polar metabolites formed by the adrenal preparations were tentatively identified as fhydroxydigitoxigenin and 168-hydroxydigitoxigenin.
INTRODUCTION The cardioactive steroid digitoxigenin 58,148-card-20(22)-enolide) lide type are biotransformed
(38,14-dihydroxy-
and other steroids of the cardenoto the pharmacologically
inactive
3a-hydroxy epimers by the 3-hydroxysteroid dehydrogenases found in rat liver (1,2,3). steps:
The epimerization takes place fn two
First, the substrate, having the 3-hydroxyl group in the
8 configuration, is oxidized to the corresponding 3-keto compound by 3B-hydroxysteroid dehydrogenase
(E.C..l.l.l.51).
The
3-keto compound is then reduced to the 3a-hydroxyl compound by 3ahydroxysteroid dehydrogenase
(E.C. 1.1.1.50).
The pyridine nuc-
leotide coenzymes apparently serve as hydrogen carriers in these steps (1,3).
STEROIDS
88
21:l
In the synthesis of steroid hormones, the conversion of pregnenolone to progesterone involves the conversion of a B-hydroxyl group to a carbonyl group at position 3 and a subsequent rearrangement of a double bond from A5 to A4. This conversion has been demonstrated -in vitro with rat ovarian tissue (4) and adrenal tissue (5). Furthermore, the conversfon of 38-hydroxypregnanesto their 3a-hydroxy epimers has been recently demonstrated in rat ovarian cytosol (6). The oxidoreductases that catalyze these conversions may al.80metabolize digitoxfgenin, To examine this possibility, we have investigated the metabolism of digitoxfgenin by rat liver, adrenal, and ovary homogenates under fdentfcal incubation conditions.
A generous supply of 3H-digitoxigeninwas provfded by Gesellschaft fur Kernforschung, M.B.H., Germany. The speciffc activity was 655 niC/mg. Thfs compound was purified by preparative thin layer chromatography (TLC) to a radfochemicaf purity of greater than 98 per cent by methods prevfousllydescribed (2). Male and fernaLerats derived from the Holtzmann strain were obtained from ,Sasco,Enc., Omaha, Nebraska and were maintained on Purina Laboratory Chow and tap water, ad libitum. A total of 35 male and 35 female rats, 255-225 gm, were used Pm these experiments. Animals were fasted overnight prior to sacrifice. All animals were sacrificed between 7:30 and 8:00 A.M. by cervical.dislocation and exsanguination. Tissue was quickly excised and immediately placed in ice-cold 0.25 M sucrose 9 TMN (5.55 M Tris chloride, pH 7.4, 0.005 M MgCP2$ and 0.010 M NaCP) buffer. Tissue pooled from 15 rats was used to prepare Ifver, ovary, and adrenal homogenates. Xn each case, tissue (1 gm/lZ ml) was homogenized in 0.25 M sucrose + TMN solution using a Potter-Elvehjem homogenizer, and 2 ml of the resulting homogenates were added to each of three 2.5ml Erlenmeyer flasks tontaining the incubation mixture. Each flask contained 1.25 ml 0.25 M sucrose + TMN buffer in which were dissolved 3.5 mg NADP+, 14 mg glucose &-phosphate, .5 units glucose &phosphate dehydrogenase, 7.3 mg nicotinamide, and 15 mg bovine serum albumfn. One mg of carrier digitoxigenin and 0.5 PC 3H-d~g~toxigeninwere added to the fncubatiou mixture Pn 0.2 ml 95% ethanol. AlI.flasks were Incubated for 30 min on an
Jan. 1973
STEROIDS
89
Eberbach water bath shaker at 37OC, gassing the flasks with 95% O2-5% C02. The incubations were stopped and 3H-digitoxigenin and metabolites were extracted with methylene dichloride and assayed by TLC and liquid scintillation counting as previously described (2). For each tissue, the per cent conversion to the digitoxigenin major metabolite was calculated, and results were expressed as nmoles product formed/min/lOO mg tissue. Student's t-test was used to compare rates obtained with female liver and adrenals to those obtained with male liver and adrenals (see Table 1). The experiment was repeated once and the results pooled, accounting for the 6 determinations/tissue indicated in Table 1. A small amount of the radioactivity in the male and female adrenal and liver homogenate extracts (0.5-2%) was localized in a low Rf band, indicating that small amounts of digitoxigenin polar metabolites had been formed during the incubations. The adrenal extracts were pooLed and concentrated to dryness. The residue was redissolved in approximately 0.5 ml of methylene dichloride-methanol (1:1), and spotted on a preparative silica gel H plate (0.5 mm thick) which was developed once in chloroform-isopropanol (9:l). The polar metabolite band, Pocalized by the method of Bhavnani (7), was removed from the plate, placed in a glass-stoppered 250 ml Erlenmeyer flask containing 50 ml methylene dichloride-methanol (1:1), and extracted 2 hr on a shaker. This procedure was found to remove all of the radioactivity from the gel. The silica gel was removed by filtration. The filtrate was concentrated to dryness, in vacua, the residue was redissolved in 2 ml of methylene dichloride-methanol (l:l), and this solution was tested by TLC, developing a silica gel II plate 4 times in chloroform-isopropanol (9:l). Upon spraying the plate with 20% ll2SO4 and heating it at llO" for 5 min, two spots of different color and about equal intensity resolved. The colors and Rf values obtained suggested to us that these polar metabolites may be 5-hydroxydigitoxigenin (periplogenin)+c and 168-hydroxydigitoxigenin (gitoxigenin).** Therefore, the extract from the purification described above was concentrated to dryness and the preparative procedure was repeated, using authentic 5-hydroxydigitoxigenin and 16+hydroxydigftoxigenin as marker steroids and removing the areas on the preparative plate (developed 4 times in chloroform-isopropanol (9:l)) corresponding to them. The two metabolites isolated (Pletabolite I and MetaboPite II> were co-chromatographed with standards on a silica gel H plate developed 4 times in the above-mentioned solvent system, Co-chromatography was also performed in a second system using silica gel I plates developed 4 times in cyclohexane-acetone-acetic acid (65:33:2): Spots were visualized with either 20% H2SO4 or anisaldehyde sp&y reagent (0.5 ml anisaldehyde, 1.0 ml cont. H2SO4, and 50 ml glacial acetic acid). Using the latter solvent system and the two spray reagents, we were previously able to distinguish between 13 digitoxigenin derivatives (8). The results of the chromatographic analyses are presented in Table 2. ,
STEROIDS
90
21:l
RESULTS The major metabolites of digitoxigenin and their rates of formation obtained with homogenates of various tissues are given in Table 1. TABLE 1 Metabolism of 'H-Digitoxlgenin by Tissue Homogenates from Male and Female Rats
Tissue*
3-Digitoxigenone formation (nmoles/min/lOOmg tissue)
3-Ep~d~g~tox~gen~n formation (nmoles/mfn/l00mg tissue)
Adrenals (male)
6.34 + 0,55
0.0
Adrenals (female)
6.99 2 0.87"
0.0
Liver
0.0
(male)
14.X.02 1.74
Liver (female)
0.0
4.40 " 0.88
Ovaries
0.0
4.20 -4_ 0.83
*n = 6 determinations 1n.s. with respect to male adrenal homogenates 2p 0.001 with respect to male liver homogenates
In liver homogenates obtained from rats of either Bex, 3-epidigitoxigenfn* was the major product. The results indicate that epimerizatfon proceeded more rapidly fn male liver homog in female liver homogenates, Epimerizatfon was also obs ovary homogenates, at a rate simflar to that observed wfth female liver homogenates. Adrenal homogenates obtained from either
91
STEROIDS
Jan. 1973
TABLE 2 Co-chromatographyof 3H-Digitoxigenin Polar Metabolites Formed by Adrenal Homogenates Obtained from Male and Female Rats
Rf
Compound(s)
Color
System 11 System XI2 H2SO4 Aniealdehyde
Metabolfte L
0.48
0.40
green
blue
5-hydroxydigitoxigenin
0.48
0.40
green
blue
5+ydroxydigitoxEgenin
0.48
0.40
green
blue
Metabolite XX
0.54
0.38
purple
green
16&-hydroxydigitoxigenin
0.54
0.38
purple
green
Hetabolite EL + 16S-hydroxydlgitoxigenin
0.54
0.38
purple
green
Metabolite I f
'silica gel H plates developed 4 times Pn chloroform-isopropanol 2(9:1) silica gel H plates developed 4 times in cyclohexane-acetone-acetic acid (65:33:2)
male or female rats primarily formed 3-digitoxigenone**with the oxidation occuring at similar rates in each sex. In addition to these metabolites, the adrenal and liver preparations obtained from rats of either sex formed small.amounts (0.5-Z% conversion) of polar digitoxigenin derivatives. The two polar metabolites formed by the adrenal homogenates were isolated
21:l
STEROIDS by preparative TLC (see experimental section) and examined by TLC in two solvent systems using two color-producing spray reagents. The Rf values and colors obtained with Metabolites I and II were identical with those obtained with authentic 5-hydroxydigitoxigenin and 16B-hydroxydigitoxigeninrespectively (Table 2).
The pro-
duction of these two metabolites, as well as several other previously unreported derivatives of digitoxigenin has been recently observed in rat liver homogenates and microsomes (Talcott and Stohs, in preparation). Furthermore, rabbit liver homogenates also produce large amounts of hydroxylated products of digitoxigenin, one of which is 5-hydroxydigitoxigenin(Bulger and Stohs, in preparation). DISCUSSION The rate-limiting step in the 3-epimerizationof cardenolides and other 3$-hydroxysteroidsby rat liver homogenates is the oxidation of the substrate to the 3-keto intermediate by 3S+ydroxysteroid dehydrogenase. This intermediate is almost immediately reduced to the epimer by 3a-hydroxysteroiddehydrogenase (1). Hence, with digitoxigenin as the substrate, appreciable amounts of 3-digitoxigenone do not accumulate in liver homogenates incubated at physiological pH (1,Z). In this study, we observed production of 3-epidigitoxigenin by ovary homogenates (Table 1) as well as by liver homogenates with no accumulation of the 3-keto intermediate. These results suggest that the epimerizing system recently described in rat ovaries (6) is similar to the system present in rat liver. In contrast, epimerization did not occur in adrenal homogenates and the major metabolite was 3-digitoxigenone. If a 3a-hydroxy-steroid
Jan. 1973
STEROIDS
dehydrogenase is present in rat adrenal homogenates, it apparently cannot reduce 3-digitoxigenoneunder our conditions. Repke and Samuels have previously observed a sex difference in 3B-hydroxysteroidepimerization and have attributed it to the "absent or exceptionally low activity" of the 3Phydroxysteroid dehydrogenase in female rat liver (1). In this regard, it is interesting that no sex difference in 3Phydroxysteroid dehydrogenase activity was noted in adrenal homogenates (see Table 1), suggesting that the liver and adrenal 3B-hydroxysteroiddehydrogenases are regulated differently. These results might be anticipated if digitoxigenin is oxidized by the same adrenal dehydrogenase that catalyzes the 3-dehydrogenationof pregnenolone piror to its conversion to progesterone. Since this step fs critical to the syntehsis of corticosteroids in both sexes, it is unlikely that 3B-hydroxysteroiddehydrogenase activity would be markedly different in male and female adrenal homogenates. The two polar derivatives of digitoxigenin produced by adrenal homogenates have been tentatively identified as 5-hydroxydigitoxigenin and 16$-hydroxydigitoxigeninby co-chromatographyof the metabolites with standards (Table 2). Rat liver homogenates also generate polar metabolites of digitoxigenin and the conversion can be greatly enhanced by prior treatment of the animals with phenobarbital (2). Preliminary evidence indicates that these polar metabolites include 5-hydroxydigitoxigeninand 16g-hydroxydigitoxPgenin plus several other derivatives, as yet unidentified, Work on positive identificationof the polar metabolites of digitoxigenin produced by rat and rabbit liver homogenates is currently in progress.
93
STEROIDS
94
An earlier finding of Repke (9) may have bearing on the pharmacodynamic significance of the results presented here. He found that 3 minutes after i.v. injection of the cardiac glycoside digitoxin, the concentration of drug found in the adrenal glands was 7-8 times that found in the liver (9). The fate of digitoxin in the adrenals is not known, but if significant amounts of it are hydroxylated on the steroid nucleus, or if the glycoside bonds can be cleaved, exposing the 38-hydroxyl group to oxidation, then the reactions which occur in adrenal homogenates with digftoxigenin as substrate may play a role in the metabolism of digitoxin.
ACKNOWLEDGEMENTS This investigation was supported in part by a National Institutes of Health Predoctoral Fellowship, #5F01 GM47428, from the National Institute of General Medical Sciences, a grant from the Nebraska Heart Association, and an Edwin Leigh Newcomb Fellowship from the American Foundation for Pharmaceutical Education.
REFERENCES 1.
Repke, K;, and Samuels, L.T., BIOCHEMISTRY 3, 689 (1964).
2.
Stohs, S.J., Reinke, L.A., and El-Olemy, M.M., BIOCHEM. PHARMACOL. 20, 473 (1971).
3.
Talcott, R.E., Stohs, S.J., and El-Olemy, M.M., BIOCHEM. PHARMACOL. 21, 2001 (1972).
4.
Huang, W.Y., and Pearlman, W.H., J. BIOL. CHEM. 237, I060 (1962).
5.
Rubin, B.L., and Dorfman, R.I., ENDOCRINOLOGY 61, 601 (1957).
6.
Nimrod, A., and Lindner, H.R., BIOCHEM. BIOPHYS. ACTA 261, 291 (1972).
7.
Bhavnani, B.R., J. CHROMATOG. 65, 454 (1972).
8.
Bulger, W.H., Talcott, R.E., and Stohs, S.J., J. CHROMATOG. 70, 187 (1972).
9.
Repke, K., ARCH. EXP. PATHOL. PHARMAKOL. 241, 165 (1961).
21:l