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The in vitro effect of 5,5′-diphenylhydantoin on the catabolism of cortisol by rat liver
The in Vitro Effect of 5,5’-Diphenylhydantoin the Catabolism of Cortisol by Rat Liver ByLEON
J. SHOLITON, EMILE
E. WERK,
The alteration of the catabolism of cortisol by rat liver slices produced by the incorporation of 5, 5’-diphenylhydantoin (DPH) in an in vitro system has been evaluated. Using appropriate control incubations, DPH markedly augmented over control values the side-chain reduction of cortisol in male rat liver incubates, whereas significant enhancement of ring-A reduction was produced in female rat liver incubates. 6-hydroxylation was decreased by DPH in both. The
JR. AND
on
JOSEPH MACGEE
major metabolite of DPH, 5-(p-hydroxyphenyl)-5-phenylhydantoin W’PH) , proved equally effective in producing these alterations. No explanation for the observed results was forthcoming from this study but it is suggested that DPH may result in the enhanced production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in rat liver slice incubates in turn augmenting the usual catabolic pathways for cortisol.
5’-DIPHENYLHYDANTOIN (DPH) in therapeutic dosage has been Shown to exert an alteration in vivo in the extra-adrenal metabolism of cortisol (F) in human subjects. 1 This effect is primarily an enhancement of the 6-hydroxylation pathway of cortisol catabolism associated with a diminution of ring-A reduction as manifested by the changes in excretion of these metabolites in urine. The purpose of the presetit study was to observe whether any alteration in the metabolism of cortisol could be produced by DPH in an in vitro system. To achieve this, a cortisol 4-Cl4 substrate and a reduced nicotinamide adenine dinucleotide phosphate (NADPH) re g enerating system were incubated with rat liver slices. Adrenal steroidogenesis in the rat differs basically from man in that its major adrenal corticosteroid is corticosterone rather than cortisol. Moreover, unlike the human subject, side-chain reduction* of cortisone has been demonstrated to be a major catabolic route in the male2s3 whereas ring-A reduction predominates in the female.3v4 However, 6-hydroxylation of cortisol does occur in rat liver incubates and, iti the male, in the same proportion which has been observed with human liver slices (unpublished data). Since increased 6-hydroxylation has been the major change ob-~ _____ ______~ ____.~~_ ~-~
5
From
the Metabolism
Section,
Veterans
Cincinnuti College of Medicine, Cincinnuti, Received for publication May 28, 1964.
Administration
Hospital
and t!~e University
of
Ohio.
*Most of the published studies which have investigated the action of rat liver on cortisol or cortisone substrates in vitro have utilized the term “side-chain” reduction in reference to C-20 hydroxylation.2*3 The side-chain reduced metabolites in this study refer to thp following compounds: Q- and /+cortol, Q- and @cortolone, 20-a OHF, 20 #l OHF, 20-aOHE and 20-fi-OHE. The ring-A reduced metabolites in the present study refer to the tetrahydro compounds, tetrahydrocortisol, allo-tetrahydrocortisol, and tetrahydrocortisone, which have been specifically isolated and quantitated but have been grouped together for presentation of data as the “ring-A reduced” fraction. 1382 METABOLISM,VOL. 13, No. 11 (NOVEMBER), 1964
IN VITRO EFFECT
OF DPH ON CATABOLISM
1383
OF CORTISOL
served with DPH in the in vivo studies cited above, rat liver slices were employed to study the action of DPH on cortisol metabolism on an in vitro basis. METHODS Preparutia
Slices:
Tissue
male and female
Wistar
of Liver
adult virgin
AND
slices rats
MATERIALS
were
obtained
(averaging
from freshly
resected
300 Gm. body weight)
livers
of
immediately
after decapitation. The animals had been fed ad libitum. Slices were cut free-hand and 1 Gm. aliquots of blotted tissue were added to each prepared incubation flask. At least 2 aliquots were obtained from each liver so that appropriate controls were available in each series of incubations. Incubation was instituted within 1 hour of sacrificing the animal. Prepmutiun
Flasks: A 0.25 PC. aliquot
of Incubation
of cortisol-4-04
in ethanol
(specific
activity, ‘70 +/mg.) was added to each flask dryness at 45 C. in a vacuum oven. The NADPH
(25 ml. Erlenmeyer) and evaporated to regenerating system was provided by the
addition
dinucleotide
of the following:
glucose-&phosphate bonate
buffer
nicotinamide
(G-6-P),
(pH 7.4)
cent solution of DPH
adenine
IO-sM.
Ten
tion. The control
of 50 ~1. of DPH
consisted
(HPPH) consisted
to pH 12)
studies
were added
diluent
alone.
(NADP)
lo-sM;
Krebs-Ringer
studies,
bicar-
50 ~1. of a 5 per
glycol, 40 per cent, and alcoto each flask prior
to incuba-
For the 5-(p-hydroxy-phenyl)-
the same concentration
of the DPH-diluent
of HPPH
was utilized;
how-
to which had been added a 30 per cent vol-
ume of 1.0 N NaOH in order to dissolve the less soluble the diluent
phosphate prepared
(2.5 mg. DPH in a diluent of propylene adjusted
ever, the diluent
of freshly
were added to each flask. For the DPH
hol, 10 per cent in water, 5-phenylhydantoin’
ml.
HPPH.
Appropriate
adjustment
of
control was also made.
Incubation: The flasks were incubated in a Dubnoff O,, 5 per cent CO,) for 5 hours at 37 C. The incubation 10 drops of glacial
shaker (atmosphere-95 per cent was terminated by the addition of
acetic acid to each flask and the incubate
frozen immediately
and stored
in a deep freeze. Extraction: which
The media
40 pg. each
solution
were thawed
had previously
been
ml. for each incubate). and partitioned
phase was extracted form extract
and washed
Extraction
(20 per cent by weight)
6-OHF,
to dryness
Separation
The
extract).
water extract
were triturated volume,
of sodium
was evaporated
3 times and the pooled
chloroform
extract
25
sulfate
The resultant
To the residual
and then extracted
to
in ethanolic
(total
addition
(3:1:12).
chloroform
was added
and Elution:
following
acetate:water
(chloroform
dium sulfate, 20 per cent by weight, give the final ethyl acetate extract.
The tissues
The ethyl acetate
heptane:ethyl
into beakers
and F standards
to dryness.
with ethyl acetate
was then performed. between
and decanted
THE,
3 times with distilled
with 160 ml. of redistilled
evaporated
Chrornutographic
THF,
added and evaporated
with a glass tissue homogenizer
dryness
at room temperature
of nonradioactive
to
polar chloro-
polar phase,
so-
with ethyl acetate
to
was chromatographed
on Whatman No. 3 paper for 20 hours in the system: toluene:methanol:water (l&3:1). The ethyl acetate extract was chromatographed for 4 hours in the system: ethyl acetate: chloroform:methanol:water ultraviolet radioactivity
light absorption with a Nuclear
strip of each chromatogram
( 1:3:2:2).
After
and marked Chicago
drying,
accordingly.
Actograph
was stained
the
chromatograms
were
viewed
for
The
chromatograms
were scanned
for
II (fig. 1 and 2).
for blue tetrazolium
Finally,
positive
a narrow
zones.
Elution
suspected end-products was accomplished with absolute methanol, subsequently to dryness and reconstituted to a volume of 0.7 ml. for each eluate.
center of all
evaporated
Quuntttotion of Metabolic End-pm&&s: A 0.25 ml. aliquot of eluate was transferred to a counting vial to which 10 ml. of toluene phosphor [4 Gm. of 2,5-diphenyloxazole and *Kindly supplied by Dr. A. Sheperdigian, Davis Co., Ann Arbor, Michigan.
Department
of Clinical
Investigation,
Parke-
1384
SHOLITON,
WERK AND MAC CEE
MALE - CONTROL
MALE - DPH
FEtiALE - CONTROL
FEMALE: DPH
On CHAIN 6 OH F MORE POLAR REbJCED UNKNOWN(S) COMPOUNDS Fig. l.-Radioscans extract DPH.
of rat liver
of representative slice
incubation
with
paired
chromatograms
a cortisol-4-Cl4
100 mg. of 1,4-b&2-( 5-phenyloxazolyl)-benzene per ing of radioactivity of each aliquot performed on spectrometer. Another 0.25 ml. aliquot of eluate was tion of the method of Silber and Porter.6 By dividing active
cortisol
THF, THE, cordingly.
metabolite
by the respective
and F were obtained
For side-chain
reduced
recovered
and the radioactivity
compounds
no recovery
of the
substrate
ethyl
with
L. of toluene] were added and counta Packard Tri-carb liquid scintillation utilized for quantitation by a modificathe known amount of added non-radioamount, counted
standards
recovery
factors
ldenrificution
of metabolites:
=
were utilized;
dpm for given fraction
X 100
dpm for given fraction
y 100
The identification
of radioactive
for &OHF,
for each eluate adjusted consequently,
results for these fractions were expressed as absolute amounts of radioactivity Radioactivity in all instances has been expressed as the per cent of cortisol-4-Cl4 per Gm. of tissue to each fraction by use of the formula: “/o conversion
acetate
and without
&OH
cortisol,
acall
detected. converted
THF,
allo-
THF, THE and F was made on the basis of the relative mob&ties of the peaks of radioactivity for these fractions as compared with the mobilities of the nonradioactive standards as determined by ultraviolet light absorption, and blue tetrazolium staining.
IN VITRO EFFECT
OF DPH ON CATABOLISM
1385
OF CORTISOL
MALE - CONTROL
MALE - DPH
.
FEMALE - CONTROL
FEMALE -‘DPH I
THE
F
\I
ALL0 THF
THF
SIDE-CHAIN REDUCED COMPOUNDS
Fig. 2.-Radioscans of representative paired chromatograms of the chloroform extract of rat liver slice incubation with a cortisol-4-W substrate with and without DPH. Since
nonradioactive
standards
for the side-chain
the identification
of these was accomplished
were tentatively
identified
chromatographs,
were added
OHF.
The
compounds
mixture
as C-20-OH
to their respective
metabolites
to a mixed
was subjected
reduced
as follows: standard
to periodic
l7-ketosteroids,
compounds
were not utilized,
Pooled samples of the eluates,
on the basis of relative consisting
of cortol,
acid oxidation,
position
cortolone,
thus converting
which were then chromatographed
which on the
and 20-
the !I&OH on What-
man no. 3 paper for 16 hours using the system: toluene:iso-octane:methyl alcohol:water 3:3:1). After drying, the strips were scanned for radioactivity. They were then stained a modified
Zimmerman
of radioactivity
stain and the standard
could then be correlated
figure 3 that the primary
side-chain
to1 but that in the chloroform
reduced
extraction
17-ketosteroids
with the respective product
thereby standards.
identified.
and 20-OHF
The peaks
It can be seen from
from these incubation
some cortolone
(1: with
studies was cor-
probably
were
also
present. Since the method described above converts both the (Y- and @position of the C20-OH to the same I7-ketosteroid it precludes separating the 20 (Y- and 20 fi- forms for identification purposes. Because of the obvious mixture of side-chain reduced metabolites in the radioactive eluates, for comparative purposes, all 20-OH compounds have been grouped together as the “side-chain reduced” fraction. Similarly, for comparison, results of conversion to THF, allo-THF, and THE were grouped together as the “ring-A reduced” fraction.
1386
SHOLJTON,
WERK AND MAC GEE
111KETO ETIOCHOLANOLONE
lbOH ETIOCHOLANOLONE
lbKETO ANMOSTENIDIONE
ll-OH
+(20-OH
E)
ANommwmlONL (lo-OH
f) ’
J
Fig. 3. -Identification of the major side-chain reduced fraction of cortisol-4-C:” after incubation with male rat liver slices. The compounds in parentheses represent the nonradioactive standards prior to oxidation to their respective keto-steroid forms which are represented in the chromatogram following the use of the modified Zimmerman stain. A more specific identification of allo-THF was also made by utilizing a similar technique. In this instance, however, a preliminary reduction with potassium borohydride was performed followed by conversion to 11-OH androsterone by periodic acid. As can be noted in figure 4, the radioactivity of the pooled eluate from female rat liver incubation would appear to be mostly allo-THF.
RESULTS Effect noted
of DPH on the Side-Chain
in table
la,
male
rat
liver
and Ring-A Reduction
preferentially
reduced
the
of Cortisol: As side-chain
of
With the addition of DPH to the incubation medium, this side reduction was augmented significantly. In table lb it can be noted, on the other hand, that with female rat slices, the major reduction route of cortisol was that of ring-A reduction with the major metabolite, in the system utilized, being allo-THF. The addition of DPH to the incubation medium resulted in significant augmentation of this pathway whereas a lesser degree of increase occurred in the proporti,on of side-chain reduced compounds. Effect of DPH on the more Polur Metabolites of Cortisol: As noted in table 1 conversion of cortisol to its 6-OH metabolite was decreased significantly by the addition of DPH to the incubation medium of Ever from both sexes. In-
cortisol.
IN VITRO EFFECT
OF DPH ON CATABOLISM
111KRTO ANDROSTlRONI r(
OF CORTISOL
1387
11-OH ANDROSTIRONE 11
II- KETO ETIOCHOL
1%OH lTlOCHOLANOLONR
Fig. 4.-Identification of the major ring-A reduced fraction of cortisol-4-C?* after incubation with female rat liver slices. The radioactive eluate was first reduced with potassium borohydride and then converted to its I7-keto form by oxidation with periodic acid. The stained chromatogram represents relative polarity of added nonradioactive standards chromatographed simultaneously. terestitigly, there was a significantly greater amount of 6-OHF produced from male than from female liver slices (5.2 f 1.3 per cent vs. 1.3 * 0.43 per cent) but notwithstanding this difference, in both sexes DPH depressed the conversion to this metabolite. In both sexes, a small radioactive peak (7.1 * 2.7 per cent in the male, 2.9 * 1.6 per cent in the female) was found in the ethyl acetate extract in a more polar position than 6-OHF. In both male and female, DPH decreased the formation of this fraction also. Effect of DPH Added after Incubation of Rat Liver and CortisoL4-C’4: To ascertain whether or not the changes noted above with DPH could be attributed to a physico-chemical effect of DPH per se in the extraction mixture rather than to an effect exerted on hepatic enzyme systems, in 3 incubations DPH was added at the standard level after incubation rather than before incubation. Figure 5 depicts the results. No deviation from the control experiments was detected. Effect of HPPH on the Catabolism of Co&sol by Rat Liver Slices: The main urinary metabolite of DPH in man, dog, and rat is 5-( p-hydroxyphenyl)-Sconjugated with glucuronic acid.6,7 Presumably phenyl hydantoin (HPPH) both the formation of the HPPH and its glucuronic acid derivati\,es occurs in the liver.6 To study the possibility that this metabohte could be the mediating agent for the changes observed in cortisol catabolism produced by DPH, 4 incubations of HPPH with appropriate controls were carried out. The results are presented in table 2. As can be noted the p-hydroxy metabohte resulted in the same basic changes as did DPH alone. Other Studies in Vitro: One or more incubations were performed utilizing possible variables in the standard incubation procedure. Adding 30,666 units of /?-glucuronidase (Ketodase, Warner-Chilcott) to the incubation media
1388
SHOLITON,
WERIC
AND
MAC
GEE
Table l.-Comparative E%ect of Diphenylhyduntoin (DPH) in Vitro on Per Cent Radioactivity of the Metubolite Fractions of Cortisol-4-C’h in Rat Liver Incubates colltro1 DPH A Mean a.
P&r
unknown
Mean
-CS.D.
Mean
?S.D.
P*
7.1
2.7
3.6
6.5
-3.6
3.2
5.2
1.3
1.2
0.46
-4.0
1.04
27.3
8.6
49.6
7.5
$22.3
4.4
2.0
4.9
3.6
+0.5
B-OHF Sideehain Ring-A b.
iS.D.
(N = IO)
Mah
reduced
reduced
6.2
2.24
zO.6
Fendee (N = 8) Polar unknown
2.9
1.6
2.0
1.1
-0.91
0.84
<0.02
6-OHF
1.3
0.43
0.81
0.31
-0.48
0.38
2.8
1.2
4.6
I .4
$1.7
2.0
24.8
8.4
49.9
15.6
+26.3
20.8
Side-chain Ring-A
lP soured
reduced
reduced
<0.02
mean differences from
t calculated
using
formula
T
=
standard
error of differences
failed to alter the changes in the proportion of cortisol metabolites produced by DPH. In several incubations to which DPH was added but in which NADP and C-6-P were not utilized, the enhancement of side-chain ring-A reduction and depression of 6OHF formation remained proportionately equal to that noted in which cofactors were added, although the absolute amount of conversion to metabolites was somewhat decreased. Lengthening the period of incubation from 5 to 18 hours only increased the relative proportion of the major reduced product (viz. side-chain reduction in the male, ring-A reduction in the female) with no effect on 6-OH cortisol formation. The DPH effect remained relatively the same. Changing the size of the incubation flask (from 25 to 125 ml.) afforded no difference. Several incubations using an atmosphere of CO?-8 per cent; N2-96 per cent: 0 2-2 per cent (relative anaerobiasis) resulted in no essential difference in the proportion of cortisol metabolites. Since the pH of the Krebs-Ringer bicarbonate buffer remained the same after the addition of the diluent and also after the incubation, pH change cannot be implicated as a source of the alteration in cortisol metabolism produced by DPH under the conditions of this study. Effect of DPH Administered in Viva on Liver Incubation: In 4 male rats DPH was administered parenterally daily for 2-4 weeks. Simultaneously for a similar period 4 other males were administered an equal volume daily of the DPH diluent to serve as controls. One of the DPH treated animals was given 5 mg. (small dose) daily for 2 weeks; another, the same daily amount for 4 weeks. One animal was given 50 mg. (large dose) daily for 2 weeks; another, the large dose daily for 4 weeks. All 8 animals were then sacrificed and liver slice incubations performed with the omission of additional diluent or DPH. As can be seen from table 3, the mean proportions of metabolite fractions remained essentially similar between control and DPH-treated animals regardless of the dose administered in vivo. DISCUSSION The
results of the present study would indicate
that the primary effect of
IN VITRO EFFECT
OF DPH ON CATABOLISM
POLAR UNKNOWN(S)
DPH
ADDED
BEFORE
DPH
ADDED
AFTER
6-OH
F
INCUBATION
INCUBATIQN
SIDE-CHAIN REDUCED COMPOUNDS
Fig. 5.-The
1389
OF CORTISOL
RING-A REDUCED COMPOUNDS
effect of DPH added before and after the incubation slices with a cortisol-4-Cl4 substrate.
of rat liver
DPH on the metabolism of cortisol by rat liver in vitro was to augment the reduction pathways through which rat liver would normally catabolize cortisol and to decrease the degree of its 6-hydroxylation. Previous investigators have pointed out the difference in catabolism of cortisol by male and female rat liver. Troop and his associate?J+* and Yates and co-workers4*s have shown that male rat liver preferentially reduces cortisone at the side-chain whereas female liver primarily reduces the ring-A. Yates et al.4 and Forchielli and co-workers lo have presented evidence that at pH 7.2 only a4-5 (u-hydrogenase (microsomal) was present in female liver whereas male liver contains the ~~-5 &hydrogenase as well. These latter investigators later reported the presence of a soluble ~~-5 fl steroid hydrogenase in female rat liver which was of a much lower activity than that of the microsomal 5 or-enzyme and had an optimum pH of about 5.6.‘l Since the incubations of the present study were performed at pH 7.4 it is therefore under-
1390
SHOLITON,
Ejject
__--_._ x
-_~~__-- 3.8
.-
~_~~.
5.6
(N=4) DPH
;
Range
(3.2-8.8) (17.2-33.2)
59.0
6.9
(4.8-10.9)
5.4
Polar unknown 6-OHF Side-chain reduced Ring-A reduced __~.~____
s
1.0
(0.6-1.2)
(50.6-67.2)
S6.4
(42.2-68.4)
(3.8-8.2)
5.4
(3.1-7.8)
(DPH) in Viuo on Per Cent of Cortisol-4-C’”
(n = 4)
-___ ;
Range
7.1 8.7 22.2 5.9
Range
__ ~1~4-$.0-1.8)-
(0.6-1.9)
1.3
Control
2
(~).2-1_6 jj-
26.8
Males
HPPH
Range
(1.4_7.0)~--1.0-
Table 3.-Comparatiue EfJect of Diphenylhydantoin Radioactivity of the Metubolite Fractions in Rat Liver Zncubates
Fractions ~_____
GEE
of DPH
Control
Polar unknown 6OHF Side-chain reduced Compds. Ring-A reduced Compds. --_____ ~____.
MAC
of the Metabolite
~~~~__
-___-__
AND
and HPPH in Vitro on the Per Cent Fractions of Cortisol-4-C’ t in Male Rat Liver Incubates --_ ____~._~_~ ~_~ __.
Table 2.-Comparatiue Radioactivity -______..
WERK
(5.2-10.3) (6.8-10.1) (20.1-25.3) (45-8.0)
~__
7.8 8.7 23.1 7.5
DPH
(n z 4) -__-
_-
Range
(5.7-11.4) (7.4-10.0) ( 18.3-25.7) (2.8-12.4)
that allo-THF has been found to be the major ring-A reduction product of female rat liver incubation. An explanation of the enhancement of these preferential routes of reduction by the presence of DPH can only be a matter for conjecture at present. It would seem logical that in any such explanation an effect on the enzymatic systems controlling cortisol catabolism must be induced. Conceivably DPH or HPPH could act in some way to stimulate the activity of the A”-reductases, 3 a-dehydrogenase, or 20 (Y- or &dehydrogenases. This might be mediated by a direct action on the enzymes or by offsetting the influence of an inhibitor of these enzymes. An attractive hypothetical explanation for the alterations in cortisol catabolism produced by DPH as noted in this study may be as follows: The work of Noach and co-workers6 and Maynert’ has shown that in the human subject and rat, HPPH is the main urinary metabolite of DPH, and that HPPH is excreted as the glucuronide after conjugation in the liver. This conjugation could result from the activation of the uridine diphosphoglucuronic acid system, the enzymes for which have been found in rat liver,‘* in the following reaction: standable
UDP-glucose
+ 2 NAD+
-f
UDP-glucuronate
+ 2H+- + 2 NADH
This reaction liberates 2 molecules of reduced nicotinamide adenine dinucleotide (NADH) which could act as hydrogen carriers for the conversion of NADP to its reduced f0rm.i” Thus, NADPH generation would be increased and in the closed system of in vitro liver incubation could allow for greater reduction of the ring-A and side-chain of the cortisol substrate.
IN VITRO EFFECT
OF DPH ON CATABOLISM
1391
OF CORTISOL
Against this concept of increased NADPH formation by DPH producing more reduced cortisol metabolites are the following facts, however. (I) With the minute amount of cortisol-4-Cl4 present as substrate in each incubation, the added cofactors, NADP and G-6-P, would generate more than adequate amounts of NADPH to completely convert the cortisol to its reduced end-products. (2) The amount of uticonverted cortisol ( <5 per cent of original radioactivity) was essentially the same for the incubates containing DPH as for the controls. Lipman et al. I4 have suggested that in human liver incubation estrone enhances the conversion of F to 6-OHF by means of a compensatory route of metabolism resulting from its depression’ of THF and THE formation. If this is so, the converse could explaiti the finding in this study of a four- to fivefold decrease in 6-OHF formation by DPH. The increase in tetrahydro formation and side-chain reduction appeared in part to be at the expense of the 6hydroxy pathway. It should be emphasized that the results of these in vitro studies are very different from those noted in in vivo studies in both the rat and human subject. It is likely that the intact animal has protective mechanisms which prevent the direct action of DPH on liver enzyme systems. Further work to elucidate these mechanisms is in progress. It would appear, however, that DPH has a profound effect on cortisol catabolism which may in some way be related to its anticonvulsant properties. ACKNOWLEDGMENT We are indebted
to Mrs. Kay Myers for her excellent
technica
assistance.
REFERENCES 1. Werk, E. E., Jr., Sholiton, L. J., and of diphenylhyMacGee, J.: Effect dantoin on cortisol metabolism. Clin. Res. 11: 230, 1963 ( abstract ) . 2. Troop, R. C.: Sex differences in metabolism of cortisone by rat liver homogenates. Fed. Proc. 17:415, 1958 Influence of gonadal hormones on the metabolism of cortisone. Endo-
crinology 64:671, 1958. 4. Yates, F. E., Herbst, A. L.,
and Urqu-
hart, J.: Sex differences in rate of ring-A reduction of Ad-3-ketosteroids in vitro by rat liver. Endocrinology 63:887,
toin. J. 122:301,
1958.
5. Silber, R. H., and Porter, C. C.: The determination of 17,21-dihydroxy-20ketosteroids in urine and plasma. J. Biol. Chem. 210:923, 1954. 6. Noach, E. L., Woodbury, D. M., and Goodman, L. S.: Studies on the absorption, distribution, fate and excre-
Pharmacol. 1958.
diphenylhydanExper.
Therap.
7. Maynert, E. W.: The metabolic fate of diphenylhydantoin in the dog, rat, and man. J. Pharmacol. Exp. Therap. 130:275, 1960. 8. Hagen,
( abstract ) . 3. -:
tion of 4-C14-labeled
A. A., and
Troop,
R.
C.:
In-
fluence of age, sex and adrenocortical status on hepatic reduction of cortisone in vitro. Endocrinology 67:194, 1960. 9. Urquhart, J., Yates, F. E., and Herbst, A. L.: Hepatic regulation of adrenal cortical function. Endocrinology 64: 816, 1959. 10. Forchielli, E., Brown-Grant, K., and Dorfman, R. I.: Steroid A4-hydrogenases of rat liver. Proc. Sot. Exp. Biol. Med. 99:594, 1958. 11. -, Ramachandrau, S., and Ringold, H. J.: The soluble steroid A4-hydrogenase of female rat liver. Steroids
1392
SHOLITON,
1: 157, 1963. 12. Strominger, J. L., Maxwell, E. S., Axelrod, J., and Kalckar, H. h4.: Enzymatic formation of uridine diphosphoglucuronic acid. J. Biol. Chem. 224: 79, 1957. 13. Villec,
C.
A.,
Hagerman,
D.
Joel, P. B.: An enzymatic the physiologic functions
1. Cortisol:
D.,
and
basis for of estro-
GLOSSAHY
OF
17a,
21-
A”-pregnene-11&
triol-3,20-dione. 2. 6-OH Cortisol:
Ad-pregnene-6/3( (Y), lla, 17a, 21-tetrol-3,20-dione. 3. Cortol: 5/3-pregnane-3a, IQ?, 17a, 2Op( a), 21-pentol. 4. Cortolone: 5fi-pregnane-3a, 1701, 2Oa
( CU),21-tetrol-II-one. 5. 20-OH
Cortisol:
n4-pregnene-II&
2Oj3 ( LY) , 2 l-tetrol-3-one. 6. Cortisone: a4-pregnene-17a, 11, 20-trione. 7. 20-OH Cortisone: 2O/j( a), 21-triol-3,
17a, 21-diol-3,
a4-pregnene-17a, 11-dione.
8. Tetrahydrocortisol: 5/3-pregnane-3a, lip, 17a, 2l-tetrol-20-one. 9. Allo-tetrahydrocortisol: 5a-pregnane-3a,
WERK AND MAC CEE
gens. In Pincus, G. (Ed. ) : Recent Progress in Hormone Research, Academic Press. New York, 1960, p. 49. 14. Lipman, M. M., Katz, F. H., and Jailer, J. W.: An alternate pathway for metabolism: B-B-hydroxycortisol cortisol production by human tis;uv slices. J. Clin. Endocrinol. 22:268, 1962. TRIVIAL NAMES
lip, 170(, 2l-tetrol-20-one. 5/3-pregnane-3a, 10. Tetrahydrocortisone: 17~u, 21-triol-11, 20-dione. A*-prenene-11/3, 2111. Corticosterone: dial3, 20-dione. 12. 11-OH Etiocholanolone: 3a, 11/I-diol-17-one.
5@androstane-
13. 11-keto Etiocholanolone: 3a-01-1 I, 17-dione.
5p-androstane-
14. 11-OH Androstenedione: lib-01-3, 17-dione.
a”-androstene-
ad-andro15. 11-keto Androstenedione: stem-3, 11, 17-trione. Androsterone: 5a-androstane16. 1 I-OH lip,
ol-17-one.
17. 11-keto Androsterone: 11, 17-dione.
5a-androstane-
Leon I. Sholiton, M.D., Stafl Physician, Veterans Administration Hospital, Cincinnati, Ohio; Assistant Professor of Medicine, University of Cincinnati College of Medicine. Emile E. Werk, Jr., M.D., Director of Medical Education, The Christ Hospital, Cincinnati, Ohio; Associate Clinical Professor of Medicine, University of Cincinnati College of Medicine. Joseph MacGee, Ph.D., Chemist, Metabolism Section, Veterans Administration Hospital, Cincinnati, Ohio; Assistant Professor of Biological Chemistry and Experimental Medicine, University of Cincinnati College of Medicine.
J. SHOLITON, EMILE
E. WERK,
The alteration of the catabolism of cortisol by rat liver slices produced by the incorporation of 5, 5’-diphenylhydantoin (DPH) in an in vitro system has been evaluated. Using appropriate control incubations, DPH markedly augmented over control values the side-chain reduction of cortisol in male rat liver incubates, whereas significant enhancement of ring-A reduction was produced in female rat liver incubates. 6-hydroxylation was decreased by DPH in both. The
JR. AND
on
JOSEPH MACGEE
major metabolite of DPH, 5-(p-hydroxyphenyl)-5-phenylhydantoin W’PH) , proved equally effective in producing these alterations. No explanation for the observed results was forthcoming from this study but it is suggested that DPH may result in the enhanced production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in rat liver slice incubates in turn augmenting the usual catabolic pathways for cortisol.
5’-DIPHENYLHYDANTOIN (DPH) in therapeutic dosage has been Shown to exert an alteration in vivo in the extra-adrenal metabolism of cortisol (F) in human subjects. 1 This effect is primarily an enhancement of the 6-hydroxylation pathway of cortisol catabolism associated with a diminution of ring-A reduction as manifested by the changes in excretion of these metabolites in urine. The purpose of the presetit study was to observe whether any alteration in the metabolism of cortisol could be produced by DPH in an in vitro system. To achieve this, a cortisol 4-Cl4 substrate and a reduced nicotinamide adenine dinucleotide phosphate (NADPH) re g enerating system were incubated with rat liver slices. Adrenal steroidogenesis in the rat differs basically from man in that its major adrenal corticosteroid is corticosterone rather than cortisol. Moreover, unlike the human subject, side-chain reduction* of cortisone has been demonstrated to be a major catabolic route in the male2s3 whereas ring-A reduction predominates in the female.3v4 However, 6-hydroxylation of cortisol does occur in rat liver incubates and, iti the male, in the same proportion which has been observed with human liver slices (unpublished data). Since increased 6-hydroxylation has been the major change ob-~ _____ ______~ ____.~~_ ~-~
5
From
the Metabolism
Section,
Veterans
Cincinnuti College of Medicine, Cincinnuti, Received for publication May 28, 1964.
Administration
Hospital
and t!~e University
of
Ohio.
*Most of the published studies which have investigated the action of rat liver on cortisol or cortisone substrates in vitro have utilized the term “side-chain” reduction in reference to C-20 hydroxylation.2*3 The side-chain reduced metabolites in this study refer to thp following compounds: Q- and /+cortol, Q- and @cortolone, 20-a OHF, 20 #l OHF, 20-aOHE and 20-fi-OHE. The ring-A reduced metabolites in the present study refer to the tetrahydro compounds, tetrahydrocortisol, allo-tetrahydrocortisol, and tetrahydrocortisone, which have been specifically isolated and quantitated but have been grouped together for presentation of data as the “ring-A reduced” fraction. 1382 METABOLISM,VOL. 13, No. 11 (NOVEMBER), 1964
IN VITRO EFFECT
OF DPH ON CATABOLISM
1383
OF CORTISOL
served with DPH in the in vivo studies cited above, rat liver slices were employed to study the action of DPH on cortisol metabolism on an in vitro basis. METHODS Preparutia
Slices:
Tissue
male and female
Wistar
of Liver
adult virgin
AND
slices rats
MATERIALS
were
obtained
(averaging
from freshly
resected
300 Gm. body weight)
livers
of
immediately
after decapitation. The animals had been fed ad libitum. Slices were cut free-hand and 1 Gm. aliquots of blotted tissue were added to each prepared incubation flask. At least 2 aliquots were obtained from each liver so that appropriate controls were available in each series of incubations. Incubation was instituted within 1 hour of sacrificing the animal. Prepmutiun
Flasks: A 0.25 PC. aliquot
of Incubation
of cortisol-4-04
in ethanol
(specific
activity, ‘70 +/mg.) was added to each flask dryness at 45 C. in a vacuum oven. The NADPH
(25 ml. Erlenmeyer) and evaporated to regenerating system was provided by the
addition
dinucleotide
of the following:
glucose-&phosphate bonate
buffer
nicotinamide
(G-6-P),
(pH 7.4)
cent solution of DPH
adenine
IO-sM.
Ten
tion. The control
of 50 ~1. of DPH
consisted
(HPPH) consisted
to pH 12)
studies
were added
diluent
alone.
(NADP)
lo-sM;
Krebs-Ringer
studies,
bicar-
50 ~1. of a 5 per
glycol, 40 per cent, and alcoto each flask prior
to incuba-
For the 5-(p-hydroxy-phenyl)-
the same concentration
of the DPH-diluent
of HPPH
was utilized;
how-
to which had been added a 30 per cent vol-
ume of 1.0 N NaOH in order to dissolve the less soluble the diluent
phosphate prepared
(2.5 mg. DPH in a diluent of propylene adjusted
ever, the diluent
of freshly
were added to each flask. For the DPH
hol, 10 per cent in water, 5-phenylhydantoin’
ml.
HPPH.
Appropriate
adjustment
of
control was also made.
Incubation: The flasks were incubated in a Dubnoff O,, 5 per cent CO,) for 5 hours at 37 C. The incubation 10 drops of glacial
shaker (atmosphere-95 per cent was terminated by the addition of
acetic acid to each flask and the incubate
frozen immediately
and stored
in a deep freeze. Extraction: which
The media
40 pg. each
solution
were thawed
had previously
been
ml. for each incubate). and partitioned
phase was extracted form extract
and washed
Extraction
(20 per cent by weight)
6-OHF,
to dryness
Separation
The
extract).
water extract
were triturated volume,
of sodium
was evaporated
3 times and the pooled
chloroform
extract
25
sulfate
The resultant
To the residual
and then extracted
to
in ethanolic
(total
addition
(3:1:12).
chloroform
was added
and Elution:
following
acetate:water
(chloroform
dium sulfate, 20 per cent by weight, give the final ethyl acetate extract.
The tissues
The ethyl acetate
heptane:ethyl
into beakers
and F standards
to dryness.
with ethyl acetate
was then performed. between
and decanted
THE,
3 times with distilled
with 160 ml. of redistilled
evaporated
Chrornutographic
THF,
added and evaporated
with a glass tissue homogenizer
dryness
at room temperature
of nonradioactive
to
polar chloro-
polar phase,
so-
with ethyl acetate
to
was chromatographed
on Whatman No. 3 paper for 20 hours in the system: toluene:methanol:water (l&3:1). The ethyl acetate extract was chromatographed for 4 hours in the system: ethyl acetate: chloroform:methanol:water ultraviolet radioactivity
light absorption with a Nuclear
strip of each chromatogram
( 1:3:2:2).
After
and marked Chicago
drying,
accordingly.
Actograph
was stained
the
chromatograms
were
viewed
for
The
chromatograms
were scanned
for
II (fig. 1 and 2).
for blue tetrazolium
Finally,
positive
a narrow
zones.
Elution
suspected end-products was accomplished with absolute methanol, subsequently to dryness and reconstituted to a volume of 0.7 ml. for each eluate.
center of all
evaporated
Quuntttotion of Metabolic End-pm&&s: A 0.25 ml. aliquot of eluate was transferred to a counting vial to which 10 ml. of toluene phosphor [4 Gm. of 2,5-diphenyloxazole and *Kindly supplied by Dr. A. Sheperdigian, Davis Co., Ann Arbor, Michigan.
Department
of Clinical
Investigation,
Parke-
1384
SHOLITON,
WERK AND MAC CEE
MALE - CONTROL
MALE - DPH
FEtiALE - CONTROL
FEMALE: DPH
On CHAIN 6 OH F MORE POLAR REbJCED UNKNOWN(S) COMPOUNDS Fig. l.-Radioscans extract DPH.
of rat liver
of representative slice
incubation
with
paired
chromatograms
a cortisol-4-Cl4
100 mg. of 1,4-b&2-( 5-phenyloxazolyl)-benzene per ing of radioactivity of each aliquot performed on spectrometer. Another 0.25 ml. aliquot of eluate was tion of the method of Silber and Porter.6 By dividing active
cortisol
THF, THE, cordingly.
metabolite
by the respective
and F were obtained
For side-chain
reduced
recovered
and the radioactivity
compounds
no recovery
of the
substrate
ethyl
with
L. of toluene] were added and counta Packard Tri-carb liquid scintillation utilized for quantitation by a modificathe known amount of added non-radioamount, counted
standards
recovery
factors
ldenrificution
of metabolites:
=
were utilized;
dpm for given fraction
X 100
dpm for given fraction
y 100
The identification
of radioactive
for &OHF,
for each eluate adjusted consequently,
results for these fractions were expressed as absolute amounts of radioactivity Radioactivity in all instances has been expressed as the per cent of cortisol-4-Cl4 per Gm. of tissue to each fraction by use of the formula: “/o conversion
acetate
and without
&OH
cortisol,
acall
detected. converted
THF,
allo-
THF, THE and F was made on the basis of the relative mob&ties of the peaks of radioactivity for these fractions as compared with the mobilities of the nonradioactive standards as determined by ultraviolet light absorption, and blue tetrazolium staining.
IN VITRO EFFECT
OF DPH ON CATABOLISM
1385
OF CORTISOL
MALE - CONTROL
MALE - DPH
.
FEMALE - CONTROL
FEMALE -‘DPH I
THE
F
\I
ALL0 THF
THF
SIDE-CHAIN REDUCED COMPOUNDS
Fig. 2.-Radioscans of representative paired chromatograms of the chloroform extract of rat liver slice incubation with a cortisol-4-W substrate with and without DPH. Since
nonradioactive
standards
for the side-chain
the identification
of these was accomplished
were tentatively
identified
chromatographs,
were added
OHF.
The
compounds
mixture
as C-20-OH
to their respective
metabolites
to a mixed
was subjected
reduced
as follows: standard
to periodic
l7-ketosteroids,
compounds
were not utilized,
Pooled samples of the eluates,
on the basis of relative consisting
of cortol,
acid oxidation,
position
cortolone,
thus converting
which were then chromatographed
which on the
and 20-
the !I&OH on What-
man no. 3 paper for 16 hours using the system: toluene:iso-octane:methyl alcohol:water 3:3:1). After drying, the strips were scanned for radioactivity. They were then stained a modified
Zimmerman
of radioactivity
stain and the standard
could then be correlated
figure 3 that the primary
side-chain
to1 but that in the chloroform
reduced
extraction
17-ketosteroids
with the respective product
thereby standards.
identified.
and 20-OHF
The peaks
It can be seen from
from these incubation
some cortolone
(1: with
studies was cor-
probably
were
also
present. Since the method described above converts both the (Y- and @position of the C20-OH to the same I7-ketosteroid it precludes separating the 20 (Y- and 20 fi- forms for identification purposes. Because of the obvious mixture of side-chain reduced metabolites in the radioactive eluates, for comparative purposes, all 20-OH compounds have been grouped together as the “side-chain reduced” fraction. Similarly, for comparison, results of conversion to THF, allo-THF, and THE were grouped together as the “ring-A reduced” fraction.
1386
SHOLJTON,
WERK AND MAC GEE
111KETO ETIOCHOLANOLONE
lbOH ETIOCHOLANOLONE
lbKETO ANMOSTENIDIONE
ll-OH
+(20-OH
E)
ANommwmlONL (lo-OH
f) ’
J
Fig. 3. -Identification of the major side-chain reduced fraction of cortisol-4-C:” after incubation with male rat liver slices. The compounds in parentheses represent the nonradioactive standards prior to oxidation to their respective keto-steroid forms which are represented in the chromatogram following the use of the modified Zimmerman stain. A more specific identification of allo-THF was also made by utilizing a similar technique. In this instance, however, a preliminary reduction with potassium borohydride was performed followed by conversion to 11-OH androsterone by periodic acid. As can be noted in figure 4, the radioactivity of the pooled eluate from female rat liver incubation would appear to be mostly allo-THF.
RESULTS Effect noted
of DPH on the Side-Chain
in table
la,
male
rat
liver
and Ring-A Reduction
preferentially
reduced
the
of Cortisol: As side-chain
of
With the addition of DPH to the incubation medium, this side reduction was augmented significantly. In table lb it can be noted, on the other hand, that with female rat slices, the major reduction route of cortisol was that of ring-A reduction with the major metabolite, in the system utilized, being allo-THF. The addition of DPH to the incubation medium resulted in significant augmentation of this pathway whereas a lesser degree of increase occurred in the proporti,on of side-chain reduced compounds. Effect of DPH on the more Polur Metabolites of Cortisol: As noted in table 1 conversion of cortisol to its 6-OH metabolite was decreased significantly by the addition of DPH to the incubation medium of Ever from both sexes. In-
cortisol.
IN VITRO EFFECT
OF DPH ON CATABOLISM
111KRTO ANDROSTlRONI r(
OF CORTISOL
1387
11-OH ANDROSTIRONE 11
II- KETO ETIOCHOL
1%OH lTlOCHOLANOLONR
Fig. 4.-Identification of the major ring-A reduced fraction of cortisol-4-C?* after incubation with female rat liver slices. The radioactive eluate was first reduced with potassium borohydride and then converted to its I7-keto form by oxidation with periodic acid. The stained chromatogram represents relative polarity of added nonradioactive standards chromatographed simultaneously. terestitigly, there was a significantly greater amount of 6-OHF produced from male than from female liver slices (5.2 f 1.3 per cent vs. 1.3 * 0.43 per cent) but notwithstanding this difference, in both sexes DPH depressed the conversion to this metabolite. In both sexes, a small radioactive peak (7.1 * 2.7 per cent in the male, 2.9 * 1.6 per cent in the female) was found in the ethyl acetate extract in a more polar position than 6-OHF. In both male and female, DPH decreased the formation of this fraction also. Effect of DPH Added after Incubation of Rat Liver and CortisoL4-C’4: To ascertain whether or not the changes noted above with DPH could be attributed to a physico-chemical effect of DPH per se in the extraction mixture rather than to an effect exerted on hepatic enzyme systems, in 3 incubations DPH was added at the standard level after incubation rather than before incubation. Figure 5 depicts the results. No deviation from the control experiments was detected. Effect of HPPH on the Catabolism of Co&sol by Rat Liver Slices: The main urinary metabolite of DPH in man, dog, and rat is 5-( p-hydroxyphenyl)-Sconjugated with glucuronic acid.6,7 Presumably phenyl hydantoin (HPPH) both the formation of the HPPH and its glucuronic acid derivati\,es occurs in the liver.6 To study the possibility that this metabohte could be the mediating agent for the changes observed in cortisol catabolism produced by DPH, 4 incubations of HPPH with appropriate controls were carried out. The results are presented in table 2. As can be noted the p-hydroxy metabohte resulted in the same basic changes as did DPH alone. Other Studies in Vitro: One or more incubations were performed utilizing possible variables in the standard incubation procedure. Adding 30,666 units of /?-glucuronidase (Ketodase, Warner-Chilcott) to the incubation media
1388
SHOLITON,
WERIC
AND
MAC
GEE
Table l.-Comparative E%ect of Diphenylhyduntoin (DPH) in Vitro on Per Cent Radioactivity of the Metubolite Fractions of Cortisol-4-C’h in Rat Liver Incubates colltro1 DPH A Mean a.
P&r
unknown
Mean
-CS.D.
Mean
?S.D.
P*
7.1
2.7
3.6
6.5
-3.6
3.2
5.2
1.3
1.2
0.46
-4.0
1.04
27.3
8.6
49.6
7.5
$22.3
4.4
2.0
4.9
3.6
+0.5
B-OHF Sideehain Ring-A b.
iS.D.
(N = IO)
Mah
reduced
reduced
6.2
2.24
zO.6
Fendee (N = 8) Polar unknown
2.9
1.6
2.0
1.1
-0.91
0.84
<0.02
6-OHF
1.3
0.43
0.81
0.31
-0.48
0.38
2.8
1.2
4.6
I .4
$1.7
2.0
24.8
8.4
49.9
15.6
+26.3
20.8
Side-chain Ring-A
lP soured
reduced
reduced
<0.02
mean differences from
t calculated
using
formula
T
=
standard
error of differences
failed to alter the changes in the proportion of cortisol metabolites produced by DPH. In several incubations to which DPH was added but in which NADP and C-6-P were not utilized, the enhancement of side-chain ring-A reduction and depression of 6OHF formation remained proportionately equal to that noted in which cofactors were added, although the absolute amount of conversion to metabolites was somewhat decreased. Lengthening the period of incubation from 5 to 18 hours only increased the relative proportion of the major reduced product (viz. side-chain reduction in the male, ring-A reduction in the female) with no effect on 6-OH cortisol formation. The DPH effect remained relatively the same. Changing the size of the incubation flask (from 25 to 125 ml.) afforded no difference. Several incubations using an atmosphere of CO?-8 per cent; N2-96 per cent: 0 2-2 per cent (relative anaerobiasis) resulted in no essential difference in the proportion of cortisol metabolites. Since the pH of the Krebs-Ringer bicarbonate buffer remained the same after the addition of the diluent and also after the incubation, pH change cannot be implicated as a source of the alteration in cortisol metabolism produced by DPH under the conditions of this study. Effect of DPH Administered in Viva on Liver Incubation: In 4 male rats DPH was administered parenterally daily for 2-4 weeks. Simultaneously for a similar period 4 other males were administered an equal volume daily of the DPH diluent to serve as controls. One of the DPH treated animals was given 5 mg. (small dose) daily for 2 weeks; another, the same daily amount for 4 weeks. One animal was given 50 mg. (large dose) daily for 2 weeks; another, the large dose daily for 4 weeks. All 8 animals were then sacrificed and liver slice incubations performed with the omission of additional diluent or DPH. As can be seen from table 3, the mean proportions of metabolite fractions remained essentially similar between control and DPH-treated animals regardless of the dose administered in vivo. DISCUSSION The
results of the present study would indicate
that the primary effect of
IN VITRO EFFECT
OF DPH ON CATABOLISM
POLAR UNKNOWN(S)
DPH
ADDED
BEFORE
DPH
ADDED
AFTER
6-OH
F
INCUBATION
INCUBATIQN
SIDE-CHAIN REDUCED COMPOUNDS
Fig. 5.-The
1389
OF CORTISOL
RING-A REDUCED COMPOUNDS
effect of DPH added before and after the incubation slices with a cortisol-4-Cl4 substrate.
of rat liver
DPH on the metabolism of cortisol by rat liver in vitro was to augment the reduction pathways through which rat liver would normally catabolize cortisol and to decrease the degree of its 6-hydroxylation. Previous investigators have pointed out the difference in catabolism of cortisol by male and female rat liver. Troop and his associate?J+* and Yates and co-workers4*s have shown that male rat liver preferentially reduces cortisone at the side-chain whereas female liver primarily reduces the ring-A. Yates et al.4 and Forchielli and co-workers lo have presented evidence that at pH 7.2 only a4-5 (u-hydrogenase (microsomal) was present in female liver whereas male liver contains the ~~-5 &hydrogenase as well. These latter investigators later reported the presence of a soluble ~~-5 fl steroid hydrogenase in female rat liver which was of a much lower activity than that of the microsomal 5 or-enzyme and had an optimum pH of about 5.6.‘l Since the incubations of the present study were performed at pH 7.4 it is therefore under-
1390
SHOLITON,
Ejject
__--_._ x
-_~~__-- 3.8
.-
~_~~.
5.6
(N=4) DPH
;
Range
(3.2-8.8) (17.2-33.2)
59.0
6.9
(4.8-10.9)
5.4
Polar unknown 6-OHF Side-chain reduced Ring-A reduced __~.~____
s
1.0
(0.6-1.2)
(50.6-67.2)
S6.4
(42.2-68.4)
(3.8-8.2)
5.4
(3.1-7.8)
(DPH) in Viuo on Per Cent of Cortisol-4-C’”
(n = 4)
-___ ;
Range
7.1 8.7 22.2 5.9
Range
__ ~1~4-$.0-1.8)-
(0.6-1.9)
1.3
Control
2
(~).2-1_6 jj-
26.8
Males
HPPH
Range
(1.4_7.0)~--1.0-
Table 3.-Comparatiue EfJect of Diphenylhydantoin Radioactivity of the Metubolite Fractions in Rat Liver Zncubates
Fractions ~_____
GEE
of DPH
Control
Polar unknown 6OHF Side-chain reduced Compds. Ring-A reduced Compds. --_____ ~____.
MAC
of the Metabolite
~~~~__
-___-__
AND
and HPPH in Vitro on the Per Cent Fractions of Cortisol-4-C’ t in Male Rat Liver Incubates --_ ____~._~_~ ~_~ __.
Table 2.-Comparatiue Radioactivity -______..
WERK
(5.2-10.3) (6.8-10.1) (20.1-25.3) (45-8.0)
~__
7.8 8.7 23.1 7.5
DPH
(n z 4) -__-
_-
Range
(5.7-11.4) (7.4-10.0) ( 18.3-25.7) (2.8-12.4)
that allo-THF has been found to be the major ring-A reduction product of female rat liver incubation. An explanation of the enhancement of these preferential routes of reduction by the presence of DPH can only be a matter for conjecture at present. It would seem logical that in any such explanation an effect on the enzymatic systems controlling cortisol catabolism must be induced. Conceivably DPH or HPPH could act in some way to stimulate the activity of the A”-reductases, 3 a-dehydrogenase, or 20 (Y- or &dehydrogenases. This might be mediated by a direct action on the enzymes or by offsetting the influence of an inhibitor of these enzymes. An attractive hypothetical explanation for the alterations in cortisol catabolism produced by DPH as noted in this study may be as follows: The work of Noach and co-workers6 and Maynert’ has shown that in the human subject and rat, HPPH is the main urinary metabolite of DPH, and that HPPH is excreted as the glucuronide after conjugation in the liver. This conjugation could result from the activation of the uridine diphosphoglucuronic acid system, the enzymes for which have been found in rat liver,‘* in the following reaction: standable
UDP-glucose
+ 2 NAD+
-f
UDP-glucuronate
+ 2H+- + 2 NADH
This reaction liberates 2 molecules of reduced nicotinamide adenine dinucleotide (NADH) which could act as hydrogen carriers for the conversion of NADP to its reduced f0rm.i” Thus, NADPH generation would be increased and in the closed system of in vitro liver incubation could allow for greater reduction of the ring-A and side-chain of the cortisol substrate.
IN VITRO EFFECT
OF DPH ON CATABOLISM
1391
OF CORTISOL
Against this concept of increased NADPH formation by DPH producing more reduced cortisol metabolites are the following facts, however. (I) With the minute amount of cortisol-4-Cl4 present as substrate in each incubation, the added cofactors, NADP and G-6-P, would generate more than adequate amounts of NADPH to completely convert the cortisol to its reduced end-products. (2) The amount of uticonverted cortisol ( <5 per cent of original radioactivity) was essentially the same for the incubates containing DPH as for the controls. Lipman et al. I4 have suggested that in human liver incubation estrone enhances the conversion of F to 6-OHF by means of a compensatory route of metabolism resulting from its depression’ of THF and THE formation. If this is so, the converse could explaiti the finding in this study of a four- to fivefold decrease in 6-OHF formation by DPH. The increase in tetrahydro formation and side-chain reduction appeared in part to be at the expense of the 6hydroxy pathway. It should be emphasized that the results of these in vitro studies are very different from those noted in in vivo studies in both the rat and human subject. It is likely that the intact animal has protective mechanisms which prevent the direct action of DPH on liver enzyme systems. Further work to elucidate these mechanisms is in progress. It would appear, however, that DPH has a profound effect on cortisol catabolism which may in some way be related to its anticonvulsant properties. ACKNOWLEDGMENT We are indebted
to Mrs. Kay Myers for her excellent
technica
assistance.
REFERENCES 1. Werk, E. E., Jr., Sholiton, L. J., and of diphenylhyMacGee, J.: Effect dantoin on cortisol metabolism. Clin. Res. 11: 230, 1963 ( abstract ) . 2. Troop, R. C.: Sex differences in metabolism of cortisone by rat liver homogenates. Fed. Proc. 17:415, 1958 Influence of gonadal hormones on the metabolism of cortisone. Endo-
crinology 64:671, 1958. 4. Yates, F. E., Herbst, A. L.,
and Urqu-
hart, J.: Sex differences in rate of ring-A reduction of Ad-3-ketosteroids in vitro by rat liver. Endocrinology 63:887,
toin. J. 122:301,
1958.
5. Silber, R. H., and Porter, C. C.: The determination of 17,21-dihydroxy-20ketosteroids in urine and plasma. J. Biol. Chem. 210:923, 1954. 6. Noach, E. L., Woodbury, D. M., and Goodman, L. S.: Studies on the absorption, distribution, fate and excre-
Pharmacol. 1958.
diphenylhydanExper.
Therap.
7. Maynert, E. W.: The metabolic fate of diphenylhydantoin in the dog, rat, and man. J. Pharmacol. Exp. Therap. 130:275, 1960. 8. Hagen,
( abstract ) . 3. -:
tion of 4-C14-labeled
A. A., and
Troop,
R.
C.:
In-
fluence of age, sex and adrenocortical status on hepatic reduction of cortisone in vitro. Endocrinology 67:194, 1960. 9. Urquhart, J., Yates, F. E., and Herbst, A. L.: Hepatic regulation of adrenal cortical function. Endocrinology 64: 816, 1959. 10. Forchielli, E., Brown-Grant, K., and Dorfman, R. I.: Steroid A4-hydrogenases of rat liver. Proc. Sot. Exp. Biol. Med. 99:594, 1958. 11. -, Ramachandrau, S., and Ringold, H. J.: The soluble steroid A4-hydrogenase of female rat liver. Steroids
1392
SHOLITON,
1: 157, 1963. 12. Strominger, J. L., Maxwell, E. S., Axelrod, J., and Kalckar, H. h4.: Enzymatic formation of uridine diphosphoglucuronic acid. J. Biol. Chem. 224: 79, 1957. 13. Villec,
C.
A.,
Hagerman,
D.
Joel, P. B.: An enzymatic the physiologic functions
1. Cortisol:
D.,
and
basis for of estro-
GLOSSAHY
OF
17a,
21-
A”-pregnene-11&
triol-3,20-dione. 2. 6-OH Cortisol:
Ad-pregnene-6/3( (Y), lla, 17a, 21-tetrol-3,20-dione. 3. Cortol: 5/3-pregnane-3a, IQ?, 17a, 2Op( a), 21-pentol. 4. Cortolone: 5fi-pregnane-3a, 1701, 2Oa
( CU),21-tetrol-II-one. 5. 20-OH
Cortisol:
n4-pregnene-II&
2Oj3 ( LY) , 2 l-tetrol-3-one. 6. Cortisone: a4-pregnene-17a, 11, 20-trione. 7. 20-OH Cortisone: 2O/j( a), 21-triol-3,
17a, 21-diol-3,
a4-pregnene-17a, 11-dione.
8. Tetrahydrocortisol: 5/3-pregnane-3a, lip, 17a, 2l-tetrol-20-one. 9. Allo-tetrahydrocortisol: 5a-pregnane-3a,
WERK AND MAC CEE
gens. In Pincus, G. (Ed. ) : Recent Progress in Hormone Research, Academic Press. New York, 1960, p. 49. 14. Lipman, M. M., Katz, F. H., and Jailer, J. W.: An alternate pathway for metabolism: B-B-hydroxycortisol cortisol production by human tis;uv slices. J. Clin. Endocrinol. 22:268, 1962. TRIVIAL NAMES
lip, 170(, 2l-tetrol-20-one. 5/3-pregnane-3a, 10. Tetrahydrocortisone: 17~u, 21-triol-11, 20-dione. A*-prenene-11/3, 2111. Corticosterone: dial3, 20-dione. 12. 11-OH Etiocholanolone: 3a, 11/I-diol-17-one.
5@androstane-
13. 11-keto Etiocholanolone: 3a-01-1 I, 17-dione.
5p-androstane-
14. 11-OH Androstenedione: lib-01-3, 17-dione.
a”-androstene-
ad-andro15. 11-keto Androstenedione: stem-3, 11, 17-trione. Androsterone: 5a-androstane16. 1 I-OH lip,
ol-17-one.
17. 11-keto Androsterone: 11, 17-dione.
5a-androstane-
Leon I. Sholiton, M.D., Stafl Physician, Veterans Administration Hospital, Cincinnati, Ohio; Assistant Professor of Medicine, University of Cincinnati College of Medicine. Emile E. Werk, Jr., M.D., Director of Medical Education, The Christ Hospital, Cincinnati, Ohio; Associate Clinical Professor of Medicine, University of Cincinnati College of Medicine. Joseph MacGee, Ph.D., Chemist, Metabolism Section, Veterans Administration Hospital, Cincinnati, Ohio; Assistant Professor of Biological Chemistry and Experimental Medicine, University of Cincinnati College of Medicine.