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Mechanism of heparin inhibition of adrenal steroid A ring reduction
Mechanism of Heparin Inhibition of Adrenal Steroid A Ring Reduction ByROBERT
C. TROOP, PATRICIA T. KRZANOWWI AND JOHN T. Brcxx,
The ability of heparin to inhibit A ring reduction of various adrenal steroids by liver homogenates has been further investigated. Protamine sulfate blocks the action of heparin on steroid metabolism and protamine by itself produces moderate inhibition of corticoid A ring reduction. Reduction of the C-17 side chain of cortisone is not inhibited by heparin. The light absorbing characteristics of TPN and TPNH are not changed in the presence of heparin which makes it unlikely that heparin binds these co-
JR.
factors of steroid reduction. Heparin had no obvious effect on the rate of conversion of TPN to TPNH by a G-6-P/G-6PD system. While the mechanism of heparin inhibition of steroid A ring reduction is still not fully known, the most likely possibility seems to be a competition between heparin and TPNH for attachment to the steroid reductases and/or a direct inhibition of the steroid reductases. (Metabolism 15: No. 6, June, 542-547, 1966)
E HAVE PREVIOUSLY PRESENTED evidence that heparin inhibits reduction of the A-‘-3-ketone group of cortisone, corticosterone, hydrocortisone and a,ldosterone by rat liver homogenates.l-” The purpose of this report is to record additional observations on this matter especially as they reiate to the possible mechanism by which heparin modifies steroid metabolism.
W
METHODS Ho&man female rats weighing between 200 and 250 Gm. were used in these studies. Details of preparing liver homogenates and of the incubation procedure have been pl>lished.2 Steroid A ring reduction was determined by the corrected ultraviolet absorption at 240 mp and C-17 side chain reduction by the Porter-Silber reaction.4 RESULTS AND DISCUSSION
Figure 1 reviews the effect cortisone reduction. Fifty per and maximum inhibition was which 2 mg. of heparin were From the Division of Clinical Memphis, Tennessee. Supported in part hy research U.S.P.H.S. Received
for publication
of various concentrations of heparin on in vitro cent inhibition occurred with 2 mg. of heparin produced by 4 mg. of heparin. Rate studies in added to the incubation system with cortisone Pharmacology,
grant Am-09151
Jan. 20, 1966.
University from
The
of Tennessee, National
Medical
Units,
lnstit~ltes of Health,
M.D.: Professor and Chairman, Division of Clinicul Phurrnucology, University of ‘Tennessee Medical Units, Memphis, Term. PATRICIA T. KRZANOWSKI, B.S.: Research Associate, Division of Clinical Pharmacology, University of Tennessee Medical Units, Memphis, Term. JOHN T. B~ccs, JR.. B.A., M.S.: Medical Student, University of Tennessee, Memphb, Tenn. ROBERT C. T~ooe,
PH.D.,
542
ADRENAL
STEROID
A RING
0
543
REDIJCl’ION
1
Milligrams
4
3
i hop&n
/
5
flask
Fig. l.-Inhibition of hepatic reduction of cortisone A ring by heparin: total volume/incubation flask was 7 ml. Per cent inhibition refers to a comparison between steroid reduction in an experimental flask and in the corresponding control flask, both prepared with the same homogenate. Each point represents the mean of 9 determinations.
and aldosterone are shown in Figures 2 and 3, respectively. Similar studies with hydrocortisone and corticosterone gave curves similar to those obtained with cortisone. The possible effect of heparin (2 mg.) on hepatic reduction of the cortisone C-17 side chain was investigated and Figure 4 shows that this parameter of steroid metabolism was not significantly modified by heparin. The reaction was measured by the phenylhydrazine-sulfuric acid method” and heparin produced a small and consistent amount of color with this reagent. When correction is made for this the curves in Figure 4 become superimposed. The fact that heparin exerts an effect on the enzyme systems responsible for steroid A ring reduction but not on those systems involved in side chain metabolism indicates that the action is somewhat specific and not simply an overall enzyme inhibition. Protamine sulfate is known to antagonize the anticoagulant properties of heparin both in vivo and in vitro and it seemed pertinent to determine whether or not protamine would also modify the effect of heparin on adrenal steroid metabolism. Three mg. of protamine antagonizes thr anticoagulant action of 100 units of heparin. Two mg. of heparin were shown, in the current study, to inhibit cortisone A ring reduction about 50 per cent. The specific activity of the hepariu emploved was 100 units/mg.. therefore, 2 mg. of heparin were studied
544
TROOP, KRZANOWSKI
AND BIGGS
0.400
a 0.300 E $ (u z iz 0.200 iii ;: 0 t O 0.100
u--o CONTROL .-. 0.000
1 0
HEPARIN,
I 2
I
I,
3
2 mg
I
4
N=4
I,,
5
6
7
TIME
8
1
I,,
9
IO II
12
,
,
,
13
14
I5
IN MINUTES
Fig. e--Effect of heparin on the rate of cortisone A ring reduction by liver homogenates. The rate of decrease in optical density is indicative of steroid A ring reduction.
0.400
3.
E
0.300
o--o
CONTROL
.-
HEPARIN,
2 mg
!z : 2
0.200
!i i 5 :
0.100
0.000
51 I
Fig. 3.-Effect homogenates.
11
2
I
I
I
I
I
I
I
I
I
I-
6
7
8
9
IO
II
12
I3
I4
I5
TIME
IN
I1
3
4
5
MINUTES
of heparin on the rate of aldosterone A ring reduction by liver
ADRENAL
STEROID
54s
A RING REDUCTION
0.400
E’ 0.300
1
0
i
0200-+~,
d 0
-----___‘-_ ------w--_-,0_
F :
0 IOO-
o,ooo
1,
,
::
I
IO
:p:::p:
2m:
3c
60 TIME
Fig. 4.-Rate of hepatic presence of heparin.
,
reduction
N=3
,
120
90
IN MINUTES
of the C-17
side chain
of cortisone
in the
in the presence of 6 mg. of protamine. In Table 1 it is seen that protamine blocks the action of heparin on A ring reduction. It is also noted that protamine sulfate by itself produces moderate inhibition of corticoid reduction and that the actions of these two substances are antagonistic. Heparin and protaminr are highly and oppositely charged molecules and these properties probably play a role in the observed effects on steroid metabolism. Table ____
l.-Eflect .~
of Protamine Sulfate on Heparin Cortisone A Ring Reduction ._ _ ____ Additions
Inhibition
of
to System
Homogenntr NO.
-_
1
0.00
0.00
197
I
2.00
0.00
72
63
1
0.00
I
2.00
6.00 6.00
155 178
“I 10
2
0.00
0.00
237
-
2 2 2 .,.,
2.00 0.00 2.00
0.00 6.00 6.00
104 196 217
56 I7 8
0.00 2.00 0.00 2.00
0.00 0.00 6.00 6.00
162 64 120 IS7
60 26 3
3 3 3
-
546
TROOP, KRZAh-OWSIiI
a
CONTROL
.-
HEPARIN,
.-w.
00 0
I
2
3 TIME
Fig. 5.-Rate
4
5 IN
6
2 mg
HEPARIN, ADDED AT
7
8
AND BICCY
9
I
2 mg MIN
10
MINUTES
of TPN reduction in the presence of heparin.
Having shown that heparin inhibits steroid A ring reduction it was important to consider the various mechanisms by which this inhibition would likely occur. The more obvious of these are: (1) Heparin could combine with the steroid molecule and protect it from reduction. This seems rather unlikely because even very large amounts of heparin failed to produce complete inhibition. (2) The availability of reduced TPN is considered to be a rate-limiting factor in hepatic A ring reduction.5 Heparin might bind either TPN or TPNH thus making these compounds unavailable for reaction. This possibility was tested by investigating the spectural characteristics (Carey spectrophotometer ) of both nucleotides in the presence and absence of heparin. If heparin binds either of the 2 forms of TPN, a change in absorption curves might be expected to occur. However, no change in the curves was noted. These experiments do not rule out the possibility that binding of TPN or TPNH by heparin does occur without a detectable change in absorption bands. (3) Heparin could inhibit the dehydrogenases responsible for reduction of TPN, in this case glucoseB-phosphate and isocitric dehydrogenase specifically; or heparin could compete with TPN for attachment to the dehydrogenases. We have previously shown2 that an excess of TPN will overcome the inhibitory effect of heparin on cortisone reduction when the substrate for TPNH production is either isoThis is suggestive of competition between citrate or glucose-6-phosphate. heparin and TPN for sites on the dehydrogenase molecules or of competition between heparin and TPNH for attachment to the steroid reductases responsible for reduction of the cortisone double bond. The most direct method for
.4.DRENAL
STEROlD
A RING
REDUCTION
54;
determining whether heparin inhibits the production of TPNH by a dehydrogenase system is to measure the rate of reduction of TPN (E 340 mp) in the presence of heparin. The system employed in this study consisted of glucose-6phosphate, phosphate buffer pH 7.4, TPN and crystalline glucose-6-phosphate dchydrogenase (Sigma). Figure 5 shows that heparin had no effect on the rate of conversion of TPN to TPNH. However, this in no way rules out the possibility of heparin competing with TPNEI for reaction sites on the steroid reductases. (4) Finally, heparin might directly inhibit the steroid reducing enzymes. Tomkins” has demonstrated that the steroid reductases exhibit substrate specificity-isolated enzymes which rapidly reduced cortisone had little effect on hydrocortisonc. If heparin did inhibit cortisone reductase specifically, it would be expected to have little effect on the other adrenal steroids. This, as we have shown, is not the case and perhaps indicates that heparin inhibits those enzymes in a rather nonspecific manner or that it produces the effect by acting on other compouents of the multienzyme system responsible for steroid A ring reduction. The possible effect of heparin directly on steroid reductases is currently being invrstigatcd in liver microsome preparations. REFERENCES 1. ‘I’roop. 1~. C.. and Siggs. J. T.: Inhibition of adrend steroid A ring reduction 1~ heparin. Program, Endocrine Socic~t\.. 1965. p. 132. 2. I-. and -: Effect of heparin on A rin:; rcdrlctiolr of adrenal steroids hv lix ~‘1 homogenates. Metabolism 14:867: 196~. 3. -. and -: Elrect of heparin 01, xlrcwal steroid mt~t;tl~olism. Escerpta \Ietlica International Congress Series iVn. 99, 1963. 1’. EHR. 4. Porter. C. C.. .jncl Silber. R. II.: A I~~GLW
titative xrd
color
steroids. .5. IIrcluhart.
J. Biol. J., Yates.
I,.: Helxttic c,oid
reaction
for
cortisone
17. 21-dihvdrosv-2O-k~,to-
related
Chem.
rrglllotion
flmctiot,.
185:201,
1950.
F. E.. ancl Herlxt. of adrenal
Endocrinolog\
A. cwtl-
64:X
16.
1959. 6. Tomkins.
(:.
of hormone
hl.:
Enzkmatic
metal~olism.
medlanism I. Oxitlatiotl-
reduction of the steroid nucleus. l’rog. Hormone Res. 123125. 1065.
Rt*c.
C. TROOP, PATRICIA T. KRZANOWWI AND JOHN T. Brcxx,
The ability of heparin to inhibit A ring reduction of various adrenal steroids by liver homogenates has been further investigated. Protamine sulfate blocks the action of heparin on steroid metabolism and protamine by itself produces moderate inhibition of corticoid A ring reduction. Reduction of the C-17 side chain of cortisone is not inhibited by heparin. The light absorbing characteristics of TPN and TPNH are not changed in the presence of heparin which makes it unlikely that heparin binds these co-
JR.
factors of steroid reduction. Heparin had no obvious effect on the rate of conversion of TPN to TPNH by a G-6-P/G-6PD system. While the mechanism of heparin inhibition of steroid A ring reduction is still not fully known, the most likely possibility seems to be a competition between heparin and TPNH for attachment to the steroid reductases and/or a direct inhibition of the steroid reductases. (Metabolism 15: No. 6, June, 542-547, 1966)
E HAVE PREVIOUSLY PRESENTED evidence that heparin inhibits reduction of the A-‘-3-ketone group of cortisone, corticosterone, hydrocortisone and a,ldosterone by rat liver homogenates.l-” The purpose of this report is to record additional observations on this matter especially as they reiate to the possible mechanism by which heparin modifies steroid metabolism.
W
METHODS Ho&man female rats weighing between 200 and 250 Gm. were used in these studies. Details of preparing liver homogenates and of the incubation procedure have been pl>lished.2 Steroid A ring reduction was determined by the corrected ultraviolet absorption at 240 mp and C-17 side chain reduction by the Porter-Silber reaction.4 RESULTS AND DISCUSSION
Figure 1 reviews the effect cortisone reduction. Fifty per and maximum inhibition was which 2 mg. of heparin were From the Division of Clinical Memphis, Tennessee. Supported in part hy research U.S.P.H.S. Received
for publication
of various concentrations of heparin on in vitro cent inhibition occurred with 2 mg. of heparin produced by 4 mg. of heparin. Rate studies in added to the incubation system with cortisone Pharmacology,
grant Am-09151
Jan. 20, 1966.
University from
The
of Tennessee, National
Medical
Units,
lnstit~ltes of Health,
M.D.: Professor and Chairman, Division of Clinicul Phurrnucology, University of ‘Tennessee Medical Units, Memphis, Term. PATRICIA T. KRZANOWSKI, B.S.: Research Associate, Division of Clinical Pharmacology, University of Tennessee Medical Units, Memphis, Term. JOHN T. B~ccs, JR.. B.A., M.S.: Medical Student, University of Tennessee, Memphb, Tenn. ROBERT C. T~ooe,
PH.D.,
542
ADRENAL
STEROID
A RING
0
543
REDIJCl’ION
1
Milligrams
4
3
i hop&n
/
5
flask
Fig. l.-Inhibition of hepatic reduction of cortisone A ring by heparin: total volume/incubation flask was 7 ml. Per cent inhibition refers to a comparison between steroid reduction in an experimental flask and in the corresponding control flask, both prepared with the same homogenate. Each point represents the mean of 9 determinations.
and aldosterone are shown in Figures 2 and 3, respectively. Similar studies with hydrocortisone and corticosterone gave curves similar to those obtained with cortisone. The possible effect of heparin (2 mg.) on hepatic reduction of the cortisone C-17 side chain was investigated and Figure 4 shows that this parameter of steroid metabolism was not significantly modified by heparin. The reaction was measured by the phenylhydrazine-sulfuric acid method” and heparin produced a small and consistent amount of color with this reagent. When correction is made for this the curves in Figure 4 become superimposed. The fact that heparin exerts an effect on the enzyme systems responsible for steroid A ring reduction but not on those systems involved in side chain metabolism indicates that the action is somewhat specific and not simply an overall enzyme inhibition. Protamine sulfate is known to antagonize the anticoagulant properties of heparin both in vivo and in vitro and it seemed pertinent to determine whether or not protamine would also modify the effect of heparin on adrenal steroid metabolism. Three mg. of protamine antagonizes thr anticoagulant action of 100 units of heparin. Two mg. of heparin were shown, in the current study, to inhibit cortisone A ring reduction about 50 per cent. The specific activity of the hepariu emploved was 100 units/mg.. therefore, 2 mg. of heparin were studied
544
TROOP, KRZANOWSKI
AND BIGGS
0.400
a 0.300 E $ (u z iz 0.200 iii ;: 0 t O 0.100
u--o CONTROL .-. 0.000
1 0
HEPARIN,
I 2
I
I,
3
2 mg
I
4
N=4
I,,
5
6
7
TIME
8
1
I,,
9
IO II
12
,
,
,
13
14
I5
IN MINUTES
Fig. e--Effect of heparin on the rate of cortisone A ring reduction by liver homogenates. The rate of decrease in optical density is indicative of steroid A ring reduction.
0.400
3.
E
0.300
o--o
CONTROL
.-
HEPARIN,
2 mg
!z : 2
0.200
!i i 5 :
0.100
0.000
51 I
Fig. 3.-Effect homogenates.
11
2
I
I
I
I
I
I
I
I
I
I-
6
7
8
9
IO
II
12
I3
I4
I5
TIME
IN
I1
3
4
5
MINUTES
of heparin on the rate of aldosterone A ring reduction by liver
ADRENAL
STEROID
54s
A RING REDUCTION
0.400
E’ 0.300
1
0
i
0200-+~,
d 0
-----___‘-_ ------w--_-,0_
F :
0 IOO-
o,ooo
1,
,
::
I
IO
:p:::p:
2m:
3c
60 TIME
Fig. 4.-Rate of hepatic presence of heparin.
,
reduction
N=3
,
120
90
IN MINUTES
of the C-17
side chain
of cortisone
in the
in the presence of 6 mg. of protamine. In Table 1 it is seen that protamine blocks the action of heparin on A ring reduction. It is also noted that protamine sulfate by itself produces moderate inhibition of corticoid reduction and that the actions of these two substances are antagonistic. Heparin and protaminr are highly and oppositely charged molecules and these properties probably play a role in the observed effects on steroid metabolism. Table ____
l.-Eflect .~
of Protamine Sulfate on Heparin Cortisone A Ring Reduction ._ _ ____ Additions
Inhibition
of
to System
Homogenntr NO.
-_
1
0.00
0.00
197
I
2.00
0.00
72
63
1
0.00
I
2.00
6.00 6.00
155 178
“I 10
2
0.00
0.00
237
-
2 2 2 .,.,
2.00 0.00 2.00
0.00 6.00 6.00
104 196 217
56 I7 8
0.00 2.00 0.00 2.00
0.00 0.00 6.00 6.00
162 64 120 IS7
60 26 3
3 3 3
-
546
TROOP, KRZAh-OWSIiI
a
CONTROL
.-
HEPARIN,
.-w.
00 0
I
2
3 TIME
Fig. 5.-Rate
4
5 IN
6
2 mg
HEPARIN, ADDED AT
7
8
AND BICCY
9
I
2 mg MIN
10
MINUTES
of TPN reduction in the presence of heparin.
Having shown that heparin inhibits steroid A ring reduction it was important to consider the various mechanisms by which this inhibition would likely occur. The more obvious of these are: (1) Heparin could combine with the steroid molecule and protect it from reduction. This seems rather unlikely because even very large amounts of heparin failed to produce complete inhibition. (2) The availability of reduced TPN is considered to be a rate-limiting factor in hepatic A ring reduction.5 Heparin might bind either TPN or TPNH thus making these compounds unavailable for reaction. This possibility was tested by investigating the spectural characteristics (Carey spectrophotometer ) of both nucleotides in the presence and absence of heparin. If heparin binds either of the 2 forms of TPN, a change in absorption curves might be expected to occur. However, no change in the curves was noted. These experiments do not rule out the possibility that binding of TPN or TPNH by heparin does occur without a detectable change in absorption bands. (3) Heparin could inhibit the dehydrogenases responsible for reduction of TPN, in this case glucoseB-phosphate and isocitric dehydrogenase specifically; or heparin could compete with TPN for attachment to the dehydrogenases. We have previously shown2 that an excess of TPN will overcome the inhibitory effect of heparin on cortisone reduction when the substrate for TPNH production is either isoThis is suggestive of competition between citrate or glucose-6-phosphate. heparin and TPN for sites on the dehydrogenase molecules or of competition between heparin and TPNH for attachment to the steroid reductases responsible for reduction of the cortisone double bond. The most direct method for
.4.DRENAL
STEROlD
A RING
REDUCTION
54;
determining whether heparin inhibits the production of TPNH by a dehydrogenase system is to measure the rate of reduction of TPN (E 340 mp) in the presence of heparin. The system employed in this study consisted of glucose-6phosphate, phosphate buffer pH 7.4, TPN and crystalline glucose-6-phosphate dchydrogenase (Sigma). Figure 5 shows that heparin had no effect on the rate of conversion of TPN to TPNH. However, this in no way rules out the possibility of heparin competing with TPNEI for reaction sites on the steroid reductases. (4) Finally, heparin might directly inhibit the steroid reducing enzymes. Tomkins” has demonstrated that the steroid reductases exhibit substrate specificity-isolated enzymes which rapidly reduced cortisone had little effect on hydrocortisonc. If heparin did inhibit cortisone reductase specifically, it would be expected to have little effect on the other adrenal steroids. This, as we have shown, is not the case and perhaps indicates that heparin inhibits those enzymes in a rather nonspecific manner or that it produces the effect by acting on other compouents of the multienzyme system responsible for steroid A ring reduction. The possible effect of heparin directly on steroid reductases is currently being invrstigatcd in liver microsome preparations. REFERENCES 1. ‘I’roop. 1~. C.. and Siggs. J. T.: Inhibition of adrend steroid A ring reduction 1~ heparin. Program, Endocrine Socic~t\.. 1965. p. 132. 2. I-. and -: Effect of heparin on A rin:; rcdrlctiolr of adrenal steroids hv lix ~‘1 homogenates. Metabolism 14:867: 196~. 3. -. and -: Elrect of heparin 01, xlrcwal steroid mt~t;tl~olism. Escerpta \Ietlica International Congress Series iVn. 99, 1963. 1’. EHR. 4. Porter. C. C.. .jncl Silber. R. II.: A I~~GLW
titative xrd
color
steroids. .5. IIrcluhart.
J. Biol. J., Yates.
I,.: Helxttic c,oid
reaction
for
cortisone
17. 21-dihvdrosv-2O-k~,to-
related
Chem.
rrglllotion
flmctiot,.
185:201,
1950.
F. E.. ancl Herlxt. of adrenal
Endocrinolog\
A. cwtl-
64:X
16.
1959. 6. Tomkins.
(:.
of hormone
hl.:
Enzkmatic
metal~olism.
medlanism I. Oxitlatiotl-
reduction of the steroid nucleus. l’rog. Hormone Res. 123125. 1065.
Rt*c.