- Email: [email protected]
Acute inhibitory action of glucocorticoids on the uptake of sulphur by bone tissue in vivo
--NOTES--
729
ACUTE Im~IBITORY ACTION OF GLUCOCOR~ICOIDS ON THE UPTAKE OF SULPHUR BY B0~E TISSUE IN VIVO Me Szizetl, E. Ezer, L. Szporn~ and G. Pekete Pharmacological Laboratories of the Chemical Works o f Gedeon R i c h t e r , L t d . B u d a p e s t , Hungax~ Received January 21, 1965 AB3TRACT
S u l p h u r u p t a k e o f t h e bones o f one week o l d c h i c k s can be s t u d i e d i n a c u t e e x p e r i m e n t s by t h e method d e s c r i b e d . 35S u p t a k e i s r e d u c e d by g l u o o c o r t i c o i d s Q The o b t a i n e d dose r e s p o n s e c u r v e s a r e s t x ~ i g h t and r u n p a z ~ l l e l . T h s p r o p o r t i o n a l i n h i b i t o r y p o t e n c y o f c o r t i s o l , p r e d n i s o l o n e and dexamethasone i s i z 2,33 : 5,42. I n t h e p a s t few y e a r s , of glucocorticoids
investizations
of the
~-~luence
on , a a c o p o l y s a c o h s ~ i d e m e t a b o l i s m
been d e a l t w i t h i n numerous s t u d i e s . first that catabolic c h e s s
have
Kowalewski 1 r e p o r t e d
could be studied satisfactorily
by the uptake of 35S labelled sulphate ions into rat bone. He furthermore proved that the deposition of radioactive sulphur in the connectxve tissue was due to the use
of
sulphate ions for the synthesis of chond~oltln sulphu~ic acid which constitutes the
basic substance of connective
tissue and collagen, He also showed that in the fractured
rat
b o n e , d u r i n g t h e p r o c e s s of h e a l i n g ,
353 u p t a k e c o u l d
be significantly inhibited with cortisone. He explains this
as t h e i n h i b i t o r y
a c t i o n of c o r t i s o n e on t h e f o r m a t i o n
of
mucopolysaocharides essential in the development of collagen fibrils and which specifically incorporate sulphates,
730
S T E R O I D S
5:5
R-haloed connective tissue metabolism in the bones of intensively growing young chickens can be clearly studied with the aid of radiosulphur as indicator. In our previous
unpublished experiments,
the results of Kowalewski et al. 2'3'4'5
t
starting
from
and Rooks et al. 6
chickens were subjected to three-weeks course of treatment with massive doses of glucocorticoids.
It was found
that the incorporation of 358 could be inhibited
by two
injections weekly for three weeks of watersoluble glucocorticoid derivatives the metabolism of which being presumably faater @ban that of the water insoluble products. Thus prednlsolone 21-hemisuccinate and 21-deoxy-21-N-methylpipsrazinyl prednlsolone have a considerable inhibitory action.
It
was therefore assumed that incorporation of sulphur studied
i n a 24 h o u r p e r i o d
glucocorticoid As r e c e n t l y
- c o u l d be i n h i b i t e d
by
acute
treatment. shown by B o s t r ~ m , B e r n s t e i n "f and W h i t e h o u s e ,
in acute in vitro
experiments,
the biosynthesis
of poly-
saocharids sulphatew in the costal cartilage was inhibited by salicylate, phenylbutazone, oxyphenbutazone,
cortisol,
cincophen and 2,4-dinitrophenol. These in vitro studies and experiments with non-steroid antlphlogistic agents which gave results similer to cortisol supported our hypothesis that the sulphur metabolism of bone tissue could acutely be influenced by glucocorticoids. MATERAALS AND METHODS Young chicks of Yellow Hungarian strain were kept on
M a y 1965
$ T E R 0 I D S
731
n o r m a l d i e t and a t 28-30°C f o r f o u r o r f i v e d a y s a f t e r a a t c h i n g . On t h e s i x t h d a y , h a l f o f t h e s t e r o i d d o s e was a d m 4 n ~ s t e r e d , s u b c u t a n e o u s l y , i n a v o l u m e o f 0 . 2 5 ml i n t h e f o r m o f a s u s p e n s i o n i n a m i x t u r e o f Tween 80 and w a t e r . C o r t i s o l , p r e d n i s o l o n e and d e x a m e t h a s o n e w e r e u s e d a s f r e e a l c o h o l s , e s t r a d i o l as b e n z o a t e , d i p r o p i o n a t e and d e o x y c o r t i c o s t e r o n e a s a c e t a t e and t e s t o s t e r o n e a s p r o p i . h a t e . The o t h e r h a l f o f t h e d o s e was a d m i n i s t e r e d by t h e same r o u t e n e x t m o r n i n g , and s i x h o u r s l a t e r t h e 35S- was i n j e c t e d s u b c u t a n e o u s l y i n d o s e s o f 50 u c p e r 100 g b o d y w e i g h t i n t h e f o r m o f a 0 . 3 % Na235S04. A g r o u p o f a n i m a l s g i v e n e q u a l v o l u m e s o f s u b c u t a n e o u s Tween 80 p l u s d i s t i l led water served as controls. Next day, the animals were killed with ether, the tibiae removed, cleaned of accompanying tissues, and dried for 24 h o u r s a t 8 0 o c . i n s t e a d of s u b j e c t i n g t h e m t o t h e l e n g t h y procedure of sulphur extraction, the dry bones were pulveri z e d i n an o r d i n a r y h o u s e h o l d m i l l , and from t h e powder o b t a i n e d , p r e p a r a t i o n s o f a p p r o x i m a t e l ~ 50 n~ e a c h w e r e mad~ i n a l u m i n i u m d i s h e s . The a c t i v i t y was m e a s u r e d w i t h 2 mg/cm endwindow GM t u b e s w i t h a n O r i o n EMG d e c i m a l s c a l e r . The a c t i v i t y o b t a i n e d was c a l c u l a t e d f o r 100 mg d r y p o w d e r .
RESULTS As d e m o n s t r a t e d i n t h e t a b l e , into
t h e b o n e s o f young c h i c k s c o u l d be s t r o n g l y
by t h e u s e o f c o r t i s o l , figure
represents
curves, flat
the incorporation
pred~solone
the linear
~afluenoed
o r d e x a m e t h a s o n e . The
sections
of the dose response
i n t h e c a s e o f low and h i g h d o s e s ,
while in the middle section,
o f 35S
there
the curve is
is a combined slope
o f b = 5 0 , 2 . The d o s e r e s p o n s e c u r v e s o f t h e t h r e e
investig-
a t e d drugs run parallel. The t a b l e failed ation
reveals
that
e v e n m a s s i v e d o s e s of s t e r o i d s
to provoke a complete inhibition
of the incorpor-
of sulphur. The maximal inhibition obtained was 76 %.
Over 60-6~ % however no dose-response relationship is found. Of the steroids devoid of glucocorticoid action, estradiol
benzoate,
estradiol
dipropionate,
deoxycortico-
]. 0 1.0
2.0
4 •0
8.0
Cortisol
Cortisol
Corti sol
Cortisol
9 9
Prednisolone 2.0
Prednisolone 4.0
x average+ s.e.m.
I0 7
Prednisolone 1.0 1.0
9
9
7 8
I0
8
7 8
I0 7
I0
7 8
9 I0
9
9
9
6 7
6 9
4
Number of control animals
8
9
9
9
7 6
I0
8
Number of treated animals 3
Prednisolone 0.5 0.5 0.5
0o25
Prednisolone 0.25 0.25
0.5 0.5
2
Dose mg/animal
Cortisol
1
Substance
Table
769+97
693+-55
2206+-119
2206+-119
2152~-I19
2206+119
807+23 1055125
1578-30
2206+119 2152.*-119 2388~82
65
68
63 6!
34
46 42
14
11 32
2388~2 2834-122
72
66
2152+119
2640~87
2640~-87
51
32 40
1788+120 2152II19 2640~$7
15 27
7
Inhibition, %
1788+111 1252"25
6
Control epm/100 mg bone
1250~-47
I!91~81
1855+68 2121+66 1915M47
721+27
887+20
1271+37
1218~0 1305-64
1518 .+-71x 919133
5
Treated cpm/100 mg bone
65
68
62
40
19
72
66
51
36
21
8
mean %
O
-4
9 6 8 9
1.0
2.0
4.0
8.0
lO 8 I0 II
I0 I0
I0
l0
I0
I0
I0
I0
!0
10 8
I0 8
9
4
I0
lO
I0
0.5
20
12 7
0.25 O. 25
Estradiol~. ~ _dlpropionate J v Testosterone 5.0 "" 20.0 Deoxyeorticosterone 20.0 ao etate
Estradlol-benzoate
I0 8
0.125 0.125
9
Prednisolone 8.0
Dexamethasone
3
2
1
1227+-29
4762+141 2614Y~0
1187~40
1163+27
685+35
634+28
881+34
643+-11
I024~41
1083+24 1599-144
1507~61 1764 '-52
677+20
5
Table continued
1093+37
6598~89 3834~-162
1093+37
1093+37
2593~60
2593~60
2593~60
2593~60
2593~60
2593+60 2040-36
2593~60 2040-36
2206+119
6
0
28 31
0
0
73
75
67
75
61
58 21
39 14
69
7
0
28 31
0
0
73
75
67
75
61
39
26
69
8
-4
m m 0
~D O~
734
S T E R O I D S
5:5
%
100 90.
80, 70~
60. 50-
/7/ //
40.
302O
/ ///
10
/ o,m5
sterone,
a25
o~
~,o
and t e s t o s t e r o n e
2,o
~o
proptonate
m~/am',-~z were
administered
massive doses to determine whether the inhibition of
in
in-
corporation of 35S is a specific action of the glucocorticoids. Only testosterone proplonate in 20 mg
doses
produced a slight Ju~hibltory action. In the case of glucocorticoids,
the following
produced a 50 % J~uhlbitlon of 35S uptake: mg; prednlsolone,
0.810 mg; dexamethasone,
relatlve potencies of 1 : 2,33 : 5,42.
doses
cortisol, 1.890 0,348 m g, wlth
May 1965
8 T E RO ID S
735
DISCUSSION
From t h e p r e c e e d i u g s e c t i o n i t
clearly
emerges t h a t
t h e d e s c r i b e d p r o c e d u r e can be a p p l i e d s a t i s f a c t o r i l ~ relatively
simply to the assay of the
and
sulphur uptake
by
c h i c k e n b o n e . I n h i b i t i o n o f s u l p h u r u p t a k e by g l u c o c o z ~ t i c o i d s can be b r o u g h t a b o u t a c u t e l y , mequired f o r i n h i b i t i o n cited in the literature, Within a certain
are c o n s i d e r a b l y lower than
range, the inhibition
cuzwes f o r t h e s t e r o i d s
doses
those
iKowalewski u s e d 50 mg c o r t i s o n e l . /
t.o t h e dose o f g l u c o c o r t i c o i d s .
Of t h e s t e r o i d s
~he g l u c o c o r t i c o l d
was i n p r o p o r t i o n
Thus t h e dose r e s p o n s e
investigated
obtained run parallel,
without glucooorticoid
s t e r o n e h a s been f o u n d t o i n h i b i t
effect,
only
testo-
t o a any e x t e n t t h e i n c o r -
p o z ~ t i o n o f s u l p h u r , No dose r e s p o n s e r e l a t i o n s h i p characteristic
t o t h e g l U C o c o r t i c o i d s , c o u l d however be
produced with t e s t o s t e r o n e . The e f f e c t i v i t y qualitatively
of g l u c o c o r t i c o i d s
parallel
with their
in this
test
runs
other pharmacological
activities 8 These results do not explain the mechanism of actlan responsible for the ~nh4_bition of sulphur uptake, Never the less sulphur uptake is an indicator ~evsallng
the
presence of sulphurization processes in the connective tissue su~tance of the bone and its reduction i~variably points to metabolic inhibition. This observation corresponds to the findin~ that glucocorticoide Inhlbit different
steps of connective tissue
metabolisms
736
S T E R O I D S
5:5
REPER~NCES i. Kowalewski, Kel PROC. SOC. EXP. B±0L. ~ED., 9f, 432 / 1958/. 2. Huble, J., ACTA ENDOCR., 25, 59 /1957/. 3. Kowalewski, K., and Gouws, P., SURG, GYN. OBST., 105 I /1951/. 3. Kowalewskl, K., and Morris,n, R. T., CANAD.J.BIOCHEM. PH~TSIOL., 35, 171 /1957/. 5. Kowalewski, K., and Lyon, R. K., SURG; FORUm, 8 11957/. 6. Rooks, V. and Dorfman, R. I. METABOLISM, 12, ~ /1963/. y. ~str~m, H., Bernstein, K., and Whitehouse, M.W., BIOCHEM. PHARM~COL., 13, No.3. /19641. 8. Pekete, G., and Szporny, L., ACTA PHYSIOL. ACAD. SCI. HUNG., 19, 57 /1961/.
729
ACUTE Im~IBITORY ACTION OF GLUCOCOR~ICOIDS ON THE UPTAKE OF SULPHUR BY B0~E TISSUE IN VIVO Me Szizetl, E. Ezer, L. Szporn~ and G. Pekete Pharmacological Laboratories of the Chemical Works o f Gedeon R i c h t e r , L t d . B u d a p e s t , Hungax~ Received January 21, 1965 AB3TRACT
S u l p h u r u p t a k e o f t h e bones o f one week o l d c h i c k s can be s t u d i e d i n a c u t e e x p e r i m e n t s by t h e method d e s c r i b e d . 35S u p t a k e i s r e d u c e d by g l u o o c o r t i c o i d s Q The o b t a i n e d dose r e s p o n s e c u r v e s a r e s t x ~ i g h t and r u n p a z ~ l l e l . T h s p r o p o r t i o n a l i n h i b i t o r y p o t e n c y o f c o r t i s o l , p r e d n i s o l o n e and dexamethasone i s i z 2,33 : 5,42. I n t h e p a s t few y e a r s , of glucocorticoids
investizations
of the
~-~luence
on , a a c o p o l y s a c o h s ~ i d e m e t a b o l i s m
been d e a l t w i t h i n numerous s t u d i e s . first that catabolic c h e s s
have
Kowalewski 1 r e p o r t e d
could be studied satisfactorily
by the uptake of 35S labelled sulphate ions into rat bone. He furthermore proved that the deposition of radioactive sulphur in the connectxve tissue was due to the use
of
sulphate ions for the synthesis of chond~oltln sulphu~ic acid which constitutes the
basic substance of connective
tissue and collagen, He also showed that in the fractured
rat
b o n e , d u r i n g t h e p r o c e s s of h e a l i n g ,
353 u p t a k e c o u l d
be significantly inhibited with cortisone. He explains this
as t h e i n h i b i t o r y
a c t i o n of c o r t i s o n e on t h e f o r m a t i o n
of
mucopolysaocharides essential in the development of collagen fibrils and which specifically incorporate sulphates,
730
S T E R O I D S
5:5
R-haloed connective tissue metabolism in the bones of intensively growing young chickens can be clearly studied with the aid of radiosulphur as indicator. In our previous
unpublished experiments,
the results of Kowalewski et al. 2'3'4'5
t
starting
from
and Rooks et al. 6
chickens were subjected to three-weeks course of treatment with massive doses of glucocorticoids.
It was found
that the incorporation of 358 could be inhibited
by two
injections weekly for three weeks of watersoluble glucocorticoid derivatives the metabolism of which being presumably faater @ban that of the water insoluble products. Thus prednlsolone 21-hemisuccinate and 21-deoxy-21-N-methylpipsrazinyl prednlsolone have a considerable inhibitory action.
It
was therefore assumed that incorporation of sulphur studied
i n a 24 h o u r p e r i o d
glucocorticoid As r e c e n t l y
- c o u l d be i n h i b i t e d
by
acute
treatment. shown by B o s t r ~ m , B e r n s t e i n "f and W h i t e h o u s e ,
in acute in vitro
experiments,
the biosynthesis
of poly-
saocharids sulphatew in the costal cartilage was inhibited by salicylate, phenylbutazone, oxyphenbutazone,
cortisol,
cincophen and 2,4-dinitrophenol. These in vitro studies and experiments with non-steroid antlphlogistic agents which gave results similer to cortisol supported our hypothesis that the sulphur metabolism of bone tissue could acutely be influenced by glucocorticoids. MATERAALS AND METHODS Young chicks of Yellow Hungarian strain were kept on
M a y 1965
$ T E R 0 I D S
731
n o r m a l d i e t and a t 28-30°C f o r f o u r o r f i v e d a y s a f t e r a a t c h i n g . On t h e s i x t h d a y , h a l f o f t h e s t e r o i d d o s e was a d m 4 n ~ s t e r e d , s u b c u t a n e o u s l y , i n a v o l u m e o f 0 . 2 5 ml i n t h e f o r m o f a s u s p e n s i o n i n a m i x t u r e o f Tween 80 and w a t e r . C o r t i s o l , p r e d n i s o l o n e and d e x a m e t h a s o n e w e r e u s e d a s f r e e a l c o h o l s , e s t r a d i o l as b e n z o a t e , d i p r o p i o n a t e and d e o x y c o r t i c o s t e r o n e a s a c e t a t e and t e s t o s t e r o n e a s p r o p i . h a t e . The o t h e r h a l f o f t h e d o s e was a d m i n i s t e r e d by t h e same r o u t e n e x t m o r n i n g , and s i x h o u r s l a t e r t h e 35S- was i n j e c t e d s u b c u t a n e o u s l y i n d o s e s o f 50 u c p e r 100 g b o d y w e i g h t i n t h e f o r m o f a 0 . 3 % Na235S04. A g r o u p o f a n i m a l s g i v e n e q u a l v o l u m e s o f s u b c u t a n e o u s Tween 80 p l u s d i s t i l led water served as controls. Next day, the animals were killed with ether, the tibiae removed, cleaned of accompanying tissues, and dried for 24 h o u r s a t 8 0 o c . i n s t e a d of s u b j e c t i n g t h e m t o t h e l e n g t h y procedure of sulphur extraction, the dry bones were pulveri z e d i n an o r d i n a r y h o u s e h o l d m i l l , and from t h e powder o b t a i n e d , p r e p a r a t i o n s o f a p p r o x i m a t e l ~ 50 n~ e a c h w e r e mad~ i n a l u m i n i u m d i s h e s . The a c t i v i t y was m e a s u r e d w i t h 2 mg/cm endwindow GM t u b e s w i t h a n O r i o n EMG d e c i m a l s c a l e r . The a c t i v i t y o b t a i n e d was c a l c u l a t e d f o r 100 mg d r y p o w d e r .
RESULTS As d e m o n s t r a t e d i n t h e t a b l e , into
t h e b o n e s o f young c h i c k s c o u l d be s t r o n g l y
by t h e u s e o f c o r t i s o l , figure
represents
curves, flat
the incorporation
pred~solone
the linear
~afluenoed
o r d e x a m e t h a s o n e . The
sections
of the dose response
i n t h e c a s e o f low and h i g h d o s e s ,
while in the middle section,
o f 35S
there
the curve is
is a combined slope
o f b = 5 0 , 2 . The d o s e r e s p o n s e c u r v e s o f t h e t h r e e
investig-
a t e d drugs run parallel. The t a b l e failed ation
reveals
that
e v e n m a s s i v e d o s e s of s t e r o i d s
to provoke a complete inhibition
of the incorpor-
of sulphur. The maximal inhibition obtained was 76 %.
Over 60-6~ % however no dose-response relationship is found. Of the steroids devoid of glucocorticoid action, estradiol
benzoate,
estradiol
dipropionate,
deoxycortico-
]. 0 1.0
2.0
4 •0
8.0
Cortisol
Cortisol
Corti sol
Cortisol
9 9
Prednisolone 2.0
Prednisolone 4.0
x average+ s.e.m.
I0 7
Prednisolone 1.0 1.0
9
9
7 8
I0
8
7 8
I0 7
I0
7 8
9 I0
9
9
9
6 7
6 9
4
Number of control animals
8
9
9
9
7 6
I0
8
Number of treated animals 3
Prednisolone 0.5 0.5 0.5
0o25
Prednisolone 0.25 0.25
0.5 0.5
2
Dose mg/animal
Cortisol
1
Substance
Table
769+97
693+-55
2206+-119
2206+-119
2152~-I19
2206+119
807+23 1055125
1578-30
2206+119 2152.*-119 2388~82
65
68
63 6!
34
46 42
14
11 32
2388~2 2834-122
72
66
2152+119
2640~87
2640~-87
51
32 40
1788+120 2152II19 2640~$7
15 27
7
Inhibition, %
1788+111 1252"25
6
Control epm/100 mg bone
1250~-47
I!91~81
1855+68 2121+66 1915M47
721+27
887+20
1271+37
1218~0 1305-64
1518 .+-71x 919133
5
Treated cpm/100 mg bone
65
68
62
40
19
72
66
51
36
21
8
mean %
O
-4
9 6 8 9
1.0
2.0
4.0
8.0
lO 8 I0 II
I0 I0
I0
l0
I0
I0
I0
I0
!0
10 8
I0 8
9
4
I0
lO
I0
0.5
20
12 7
0.25 O. 25
Estradiol~. ~ _dlpropionate J v Testosterone 5.0 "" 20.0 Deoxyeorticosterone 20.0 ao etate
Estradlol-benzoate
I0 8
0.125 0.125
9
Prednisolone 8.0
Dexamethasone
3
2
1
1227+-29
4762+141 2614Y~0
1187~40
1163+27
685+35
634+28
881+34
643+-11
I024~41
1083+24 1599-144
1507~61 1764 '-52
677+20
5
Table continued
1093+37
6598~89 3834~-162
1093+37
1093+37
2593~60
2593~60
2593~60
2593~60
2593~60
2593+60 2040-36
2593~60 2040-36
2206+119
6
0
28 31
0
0
73
75
67
75
61
58 21
39 14
69
7
0
28 31
0
0
73
75
67
75
61
39
26
69
8
-4
m m 0
~D O~
734
S T E R O I D S
5:5
%
100 90.
80, 70~
60. 50-
/7/ //
40.
302O
/ ///
10
/ o,m5
sterone,
a25
o~
~,o
and t e s t o s t e r o n e
2,o
~o
proptonate
m~/am',-~z were
administered
massive doses to determine whether the inhibition of
in
in-
corporation of 35S is a specific action of the glucocorticoids. Only testosterone proplonate in 20 mg
doses
produced a slight Ju~hibltory action. In the case of glucocorticoids,
the following
produced a 50 % J~uhlbitlon of 35S uptake: mg; prednlsolone,
0.810 mg; dexamethasone,
relatlve potencies of 1 : 2,33 : 5,42.
doses
cortisol, 1.890 0,348 m g, wlth
May 1965
8 T E RO ID S
735
DISCUSSION
From t h e p r e c e e d i u g s e c t i o n i t
clearly
emerges t h a t
t h e d e s c r i b e d p r o c e d u r e can be a p p l i e d s a t i s f a c t o r i l ~ relatively
simply to the assay of the
and
sulphur uptake
by
c h i c k e n b o n e . I n h i b i t i o n o f s u l p h u r u p t a k e by g l u c o c o z ~ t i c o i d s can be b r o u g h t a b o u t a c u t e l y , mequired f o r i n h i b i t i o n cited in the literature, Within a certain
are c o n s i d e r a b l y lower than
range, the inhibition
cuzwes f o r t h e s t e r o i d s
doses
those
iKowalewski u s e d 50 mg c o r t i s o n e l . /
t.o t h e dose o f g l u c o c o r t i c o i d s .
Of t h e s t e r o i d s
~he g l u c o c o r t i c o l d
was i n p r o p o r t i o n
Thus t h e dose r e s p o n s e
investigated
obtained run parallel,
without glucooorticoid
s t e r o n e h a s been f o u n d t o i n h i b i t
effect,
only
testo-
t o a any e x t e n t t h e i n c o r -
p o z ~ t i o n o f s u l p h u r , No dose r e s p o n s e r e l a t i o n s h i p characteristic
t o t h e g l U C o c o r t i c o i d s , c o u l d however be
produced with t e s t o s t e r o n e . The e f f e c t i v i t y qualitatively
of g l u c o c o r t i c o i d s
parallel
with their
in this
test
runs
other pharmacological
activities 8 These results do not explain the mechanism of actlan responsible for the ~nh4_bition of sulphur uptake, Never the less sulphur uptake is an indicator ~evsallng
the
presence of sulphurization processes in the connective tissue su~tance of the bone and its reduction i~variably points to metabolic inhibition. This observation corresponds to the findin~ that glucocorticoide Inhlbit different
steps of connective tissue
metabolisms
736
S T E R O I D S
5:5
REPER~NCES i. Kowalewski, Kel PROC. SOC. EXP. B±0L. ~ED., 9f, 432 / 1958/. 2. Huble, J., ACTA ENDOCR., 25, 59 /1957/. 3. Kowalewski, K., and Gouws, P., SURG, GYN. OBST., 105 I /1951/. 3. Kowalewskl, K., and Morris,n, R. T., CANAD.J.BIOCHEM. PH~TSIOL., 35, 171 /1957/. 5. Kowalewski, K., and Lyon, R. K., SURG; FORUm, 8 11957/. 6. Rooks, V. and Dorfman, R. I. METABOLISM, 12, ~ /1963/. y. ~str~m, H., Bernstein, K., and Whitehouse, M.W., BIOCHEM. PHARM~COL., 13, No.3. /19641. 8. Pekete, G., and Szporny, L., ACTA PHYSIOL. ACAD. SCI. HUNG., 19, 57 /1961/.