Sulfation of corticosteroids by the adrenal of the human newborn

765

SULFATXON OF CORTTCOSTEROIDS BY THE ADRENAL OF THE HUMAN NEWBORN G. P, Klein and C. J. P. Gtroud Department of I n v e s t i g a t i v e Medicine, HcGtli U n i v e r s i t y and the Endocrine Research Laboratory, The Montreal Chtldrenms Hospital, Montreal, Canada.

Received April 2, 1965 ABSTRACT Ev|dence i s presented that adrenal s l i c e s from newborn |nfants form the C2I ester sulfates of several steroids of the pregn-q-ene-3,20-d|one s e r i e s . Radtocheratcal p u r i t y of the products was assessed by double isotope assay. The greatest y i e l d (62~) was that of 11~,21-d|hydroxypregn-q-ene-3,20--dtone-21-sulfate from c o r t i c o s t e r o n e . Hydroxylation of C21 s t e r o i d sulfates when used as precursors was not observed. The results are i n t e r p r e t e d as i n d i c a t i n g t h a t , ~f i t occurs ~n vtvo, s u i f a t t o n of such compounds may represent an ultimate reaction endtng the process of b i o s y n t h e s i s . XNTROilJCTION Sulfokinase a c t i v i t y toward a number of steroids of the pregn/4-ene-3,20-dtone series has been reported tn normal human l i v e r and adrenals ( I - 3 ) as well as tn one instance of an adrenal tumor (/4).

In the case of the adrenals of the human newborn there |s

evidence that the formation of c o r t t c o s t e r o t d C21 sulfates |s dependant upon s p e c i f i c suifokinases ( 5 ) .

The f i n d i n g of such

conjugates in the ur|ne of normal human adults and of newborn i n f a n t s (6,7) and the report of the possible secretion of corttcosterone sulfate by the human adrenal (8) has indicated the need for i n v e s t | g a t i n g to what extent s u | f a t i o n of C21 corticostero~ds takes part in s t e r o i d b~ogenesis. £XPERIMENTAL _P.reparation of Reference Stero|d..Su|fates

766

a)

b)

S T E R O I D S

Non-Radioactive: These were prepared by a modification of the method of $obel and Spoerri (9) by adding pyridine sulfur t r i o x i d e (I00 mg) to a steroid alcohol (I00 mg) in pyridine (3 ml) and by allowing the reaction to take place at room temperature for 60 hrs. The steroid sulfate formed was f i r s t separated from the corresponding unesterified steroid alcohol by thin layer chromatography on s i l i c a gel G (with chloroform-methanol-water: 8:2:0.I v/v; in this system the Rf values of the steroid sulfates studied here range from 0.15 to 0.26; the Rf of the corresponding free steroids being 1.0), crystallized from ethanol:ether and characterized by their infrared spectrum (Perkin-Elmer Model 21, in KBr p e l l e t s ) . In each instance the two main bands of absorption of the sulfate were found between 1260-1240 cm-I and I030-I025 cm- l . The presence of the sulfate ion was also assessed by the rhodizonic acid (lO) and the methylene blue ( I I ) reactions. A positive soda fluorescence reaction and a negative blue t e t r a zolium reaction provided supporting evidence for the presence of the sulfate ester at C21. Radioactive: Following sulfation, 3H or ]4C steroids were subjected to thin layer chromatography (see above). Their radiochemical purity was assessed by carrier d i l u t i o n with the appropriate non-radioactive steroid sulfate. This i n volved chromatography on the same thin layer system and on paper in systems I and I l l of Pasqualini (12). After each chromatography the specific a c t i v i t y of the material was determined by counting of the r a d i o a c t i v i t y and by either the Porter-Silber (13) or the i z o n i c o t i n i c acid hydrazide reaction (14), as applicable. I f within experimental errors, the three successive specific a c t i v i t i e s were identical, the radioactive steroid sulfate was considered as pure.

Incubation of Adrenal Slices Adrenal glands from a f u l l t e m newborn infant (age= 4 hours, weight: 2900 gm) and a premature infant (time of gestation" 24 weeks, age: 48 hours, weight= 565 gin) were obtained within four hours of death and processed prior to incubation, as prevlously described (15). No attempt was made to separate the foetal c o r t e x from the a d u l t

cortex.

Adrenal slices (250-300 mg/flask) were preincubated for 30 minutes with 15 ~d~ole of ATP disodium salt (Sigma Chem. Co.), in Krebs-Ringer bicarbonate buffer (14 ml/gm of tissue) pH 7.4., omitting CaCI2 (]6), following which the tissue and media were transferred to a new set of flasks each containing a different radioactive steroid precursor, previously dried under nitrogen. The incubation was carried out for two hours. The preincubation and incubation were performed in a Dubnoff incubator, at 37°C in an atmosphere of 02-C02 ~/o. Precursors were tracers of ],2-3H-ll-deoxycorticosterone (2 l-hydroxypregn-4-ene-3,20-dione ), 1,2-3~r-cor ti costerone ( I I~,21 -di hydroxypregn-4-ene-3,20-di one ), 1,2-3H-] ]-deoxycorti sol (lT~,2]-dihydroxypregn-4-ene-3,20-dione) and ],2-3H-cortisol

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J u n e 1965

S T E R O I D S

( l l ~ , 1 7 ~ , 2 1 - t r l h y d r o x y p r e g n - 4 - e n e - ~ p 2 0 - d i o n e ) , the s u l f a t e s of 4-14C-deoxycorticosterone and 4- C - ] l - d e o x y c o r t t s o l and in one instance 4-14C-progesterone (pregn-4-ene-3,20-dtone). E x t r a c t i o n and I d e n t i f i c a t i o n At the completion of the i n c u b a t i o n the t i s s u e was homogenized in the i n c u b a t i o n media and the p r o t e i n s p r e c i p i t a t e d w i t h two volumes of acetone-ethanol ( l : l ) . This was then f i l t e r e d , through Whatman NO.2o f i l t e r paper and the f i l t r a t e evaporated to dryness. The dry residue was redissolved in d i s t i l l e d w~ter and the s o l u t i o n e x t r a c t e d : f i r s t three times w i t h three volumes of dichloromethane, then twice w i t h one volume of n-butanol (conjugated f r a c t i o n ) . Following a d d i t i o n of 25 to 50 ~gm of appropriate non-radioa c t i v e C21 s t e r o i d s u l f a t e c a r r i e r s ( i . e . l l - d e o x y c o r t i c o s t e r o n e s u l f a t e and c o r t l c o s t e r o n e s u l f a t e in the experiment where l l deoxycorticosterone had been used as precursor), the butanol ext r a c t was subjected to t h i n layer chromatography (see above) f o r p u r i f i c a t i o n of the s u l f a t e f r a c t i o n . In t h i s system, such glucuronides as t h a t of 30(,17,21-trihydroxy-5~-pregnan-ll,20dione and of 3~-hydroxyandrost-5-ene-17-one do not m i g r a t e . Pre]imtnary separation of s t e r o i d s u l f a t e s was achieved on the system I I I of Pasqualini ( 1 2 ) . On t h i s chromatogram r a d i o a c t i v e areas were located by scanning (Nuclear Chicago Actigraph I I . ) . Those corresponding to the u l t r a v i o l e t absorbing areas of the c a r r i e r s were e l u t e d . Tol~hese f r a c t i o n s was added a t r a c e r amount of the appropriate model " C - c o r t i c o s t e r o t d s u l f a t e . Each f r a c t i o n was then p u r i f i e d to constant isotope r a t i o s by at least two paper chromatographtes of the s t e r o i d as s u l f a t e in system I I I and I (12), followed by enzyme h y d r o l y s i s (Helicase:500 u n i t sulfatase/m] of O.2 M acetate b u f f e r , pH 5.2, incubated f o r 48 h r s . at 37°C) and sequential chromatography of the free s t e r o i d and of i t s acetate in an appropriate ~/stem ( 1 7 ) . In some instances ( i n the case of l l - d e o x y c o r t i c o s t e r o n e , c o r t i c o s t e r o n e and l l - d e o x y c o r t i s o ] s u l f a t e s ) , f o l l o w i n g the f i r s t paper chromatography in system I I I , an a l i q u o t of the s t e r o i d s u l f a t e was taken and i t s radiochemica] p u r i t y e s t a b l i s h ed by carrier d i l u t i o n as described above (see: Experimental, paragraph:b). When progesterone was used as a precursor the conjugated f r a c t i o n was subjected to t h i n layer chromatography, followed by one s i n g l e paper chromatography in system I I I . On t h i s chromatogrem two r a d i o a c t i v e areas d i s t i n c t but of c l o s e l y related p o l a r i t y , were detected. The larger and more polar corresponded to the m o b i l i t y of c o r t i c o s t e r o n e and l l - d e o x y c o r t i s o l s u l f a t e s , the less polar to t h a t of l l - d e o x y c o r t i costerone s u l f a t e . These two areas were eluted together and an a l i q u o t subjected to enzyme h y d r o l y s i s as p r e v i o u s l y desc r i b e d . A f t e r adding a t r a c e r of t r t t i a t e d l l - d e o x y c o r t i costerone, c o r t t c o s t e r o n e and l l - d e o x y c o r t i s o ] free alcohol the s t e r o i d s were i d e n t i f i e d by constant isotope r a t i o through one chromatography of the Free s t e r o i d s and two chromatographies after acetylation.

767

768

S T E R 0 I D S

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T r i t i u m and I4C counts were performed i n a Packard T r i c a r b Liquid Scintillation Spectrometer (3H and 14C e f f i c i e n c y 20 and 50% r e s p e c t i v e l y ) at one s i n g l e v o l t a g e , u s i n g a m o d i f i c a t i o n of the d i s c r i m i n a t o r r a t i o method of Okita ( 1 8 ) . Steroid sulfates were r e d i s s o i v e d i n 0.2 ml of methanol p r i o r to the a d d i t i o n of iO ml o f t o l u e n e - p h o s p h o r s o l u t i o n ( 1 7 ) C o r r e c t i o n s were made f o r q u e n c h i n g . Constant i s o t o p e r a t i o s were i n t e r p r e t e d as a c r i t e r i o n of i d e n t i t y of the unknown w i t h the l a b e l l e d r e f e r e n c e s t e r o i d C2I s u l f a t e , RESULTS AND DISCUSSION The total amount of b u t a n o l - e x t r a c t a b l e - r a d i o a c t i v i t yexpressed as % of the r a d i o a c t i v i t y of the precursor added was" for

I I - d e o x y c o r t i c o s t e r o n e as p r e c u r s o r

32

f o r c o r t i costerone

77

for

58

I l-deoxycortisol

for corti sol

19

and f o r progesterone

46 %

These figures do not necessarily represent solely steroid sulfates since dichloromethane would not extract certain polar but unconjugated steroids.

From some of the precursors used

the formation of such compounds cannot be excluded (19). ~c

Upon p u r i f i c a t i o n of these extracts several steroid sulfates were characterized (Table I ) .

From corticosterone

the y i e l d of corticosterone sulfate (62%) was far greater than that of any s u l f a t e from any other precursor.

In the case of

I]-deoxycorticosterone and ]]-deoxycortiso] the y i e l d of the corresponding sulfates greatly exceeded that from t h e i r respective ]I~-hydroxylated conversion product.

In the instance

of progesterone the y i e l d of ]]-deoxycorticosterone, I]-deoxyc o r t i s o l and corticosterone sulfates decreased in that order. When I]-deoxycorticosterone sulfate or ] l - d e o x y c o r t i s o l

June 1965

S T E R O I D S

769

TABLE I Double Isotope Assay of Cortfcosterofd C21 Sulfates Produced by Human Newborn Adrenal Sltces Precursor Spectftc

Added

Actlv|ty:

3H/tkC ratios after sequential I~pe r chromatography of the steroid

i~c/mpZ4

~g

Product

OOCx~ 2.18

0.27

OOC-SO3" B-SO3-

8 .b, 2.5

a. ! 1-7

8.3 I .b,

8.3 i -5

20.8 0.8

B

0.23

B-SO3-

17.5

19.3

17.8

17.5

62 .I

S 0. ~

O.~7

S-SO3F-SO3-

13.7 2.0

13.8 2. I

14.2 I .7

14.8 I .7

30.2 I .8

F

0.23

F-SO3-

7.2

7.2

7.7

7.2

9.9

13.10

00C-SO~B-SO~" S-SO3-

-

-

5• I 28.3 2.2

5.1 28.3 2.!

8.7 I °5 h .3

1.88

C21-SO3"

Yield from Precursor x

C21-S03" C21-OH C21-OAc.

1.65 PROG. 0.02

The ytelds of individual steroid sulfates are corrected for loss from the time at which the labelled model steroids were added (see t e x t ) . In the experiment with progesterone the isotope ratios obtained after a second chromatography of the steroid acetates were: 5.1, 28.b, and 2.1 respectively. x~ See Reference (23) for these abbreviations°

sulfate

w e r e used as p r e c u r s o r s

cort|$ol

sulfates

keeping with differs

were f o r m e d .

the previous

series

of

that

can be f u r t h e r

might

if

the presence of of

prey:able

ion at

C2I

experiment

terminates

are

~aulieu ( 4 ) .

occurs

corticosterone

foetuses

The y i e l d s

it

of

of

sulfate

in

(4)j

by t h e a d r e n a l s

intermediates

in vivo,

perfused with

in

the pregn-5-

hydroxylated

as b i o s y n t h e t i c

that,

which is

nor

Lebeau and B a u l i e u

steroids

( 2 0 ) o r used i n v i v o indicate

corticosterone

This findingp

report

from the observation

en-3~-suifate

neither

(21).

This

as s u g g e s t e d by the adrenal

progesterone

(22)~

extract sulfat-

the process of biosynthesis.

steroid

comparable

sulfates to

observed in the

those reported

present

by Lebeau and

Although s l m i l a r date were obtained by us upon

770

S T E R O I D S

incubation of adrenals obtained from a premature infant (foetal age: 24 weeks) when ATP was not added, the y i e l d of steroid sulfates from i n d i v i d u a l precursors was in t h i s case substanti a l l y lower.

Therefore the higher yields obtained by Lebeau,

Baulieu and by us in the present experiment are most l i k e | y dependant on the formation of the active sulfate (adenosine3'-phosphate-St-phosphosulfate) from the ATP added to the i n cubation media. To our knowledge incubation of adrenal slices from normal adult subjects have not been performed under the conditions described here. ACKN0w ' VLEDGEMENTS The authors wish to express t h e i r gratitude to Dr. J.S.L. Browne for his i n t e r e s t and support, to Dr. F.W. Wiglesworth, Dr. J.S. Fawcett and Dr. L. Stern for providing biological material, to Drs. R. Deghenghi and G. Paplneau-Couture for t h e i r c r i t i c a l appraisal of infrared analysis, to Or. J. Stachenko and Dr. D.A. Hillman for advice and to Dr. C.W. Murphy (Ciba Co. Ltd.) for a g i f t of ll-deoxycorticosterone. This work was supported by a grant from the Medical Research Council of Canada.

Claude J. P. Giroud, M.D., Medical Research Associate of the Medical Research Council of Canada.

REFERENCES I.

Wengle, B., ACTA SOC. MED. UPSAL., 69, 105 (1964).

2.

Bostrom, H., Franksson, C. and Wengle, B., ACTA ENDOCR. (Kobenhavn) 47, 633 (1964).

3.

Adams, J. B., BIOCHIM. BIOPHYS. ACTA, 82, 572 (1964).

4.

Lebeau, M. C. and Baulieu, E. E., ENDOCRINOLOGY, 73, 832 (1963).

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June 1965

.

.

.

8.

S T E R O I D S

771

Klein, G. P., Giroud, C. J. P. and Browne, J. $. L., FED. PROC., 24, 574 (1965).

Pasqualini, J. R. and Jayle, M. F., BIOCHEM. J., 81, 147 (1961). Drayer, N. M. and Giroud, C. J. P., STEROIDS, 5, 289 (1965). Lebeau, M. C. and Baulieu, E. E., C. R. ACAD. SCI. PARIS,

258, 6265 (1964). .

Sobel, A. and Spoerri, P. E., J. AMER. CHEM. SOC., 63, 1259 (1941).

10.

Burman, D. P., ANAL. CHIM. ACTA, 9, 513 (1953).

II.

Crepy, 0., REV. FRANC. ETUD. CLIN. BIOL., 5, 283 (1960).

12.

Pasqualini, J. R., Zelnik, R. and Jayle, M. F., EXPERIENTIA, 16, 317 (1960).

13.

Porter, C. C. and Silber, R. H., J. BIOL. CHEM., 185, 201 (195o).

14.

Umberger, J. E., ANAL. CHEM., 27, 768 (1955).

15.

Hillman, D. H., Stachenko, J. and Giroud, C. J. P., THE HUMANADRENALCORTEX, Editors A. R. Currie, T. Symington and J° K. Grant, E. and S. Livingstone Ltd., London, 1962, p. 586.

16.

Umbreit, W. W., Burris, R. H. and Stauffer, J. F. MANOMETRIC TECHNIQUES, Burgess Publishing Co., Minneapolis, 1959, p. 149.

17.

Stachenko, J., Laplante, C. and Giroud, C. J. P., CANAD. J. BIOCHEM., 42, 1275 (1964).

18.

Okita, G. I . , Kabara, J. J., Richardson, F. and Leroy, G.V., NUCLEONICS, 15, 111 (1957).

19.

Touchstone, J. C., Kasparow, M. and Rosenthal, 0., FED. PROC., 18, 340 (1959).

20.

Calvin, H. I . and Lieberman, S., BIOCHEMISTRY(Wash.), 3, 259 (1964).

21.

Calvin, H. I . , Vande Wiele, R. L., and Lieberman, S., BIOCHEMISTRY (Wash.), 2, 648 (1963).

22.

Bird, C. E., W|lson, R., Wiqulst, N., Oiczfalusy, E. and Solomon, S., CANAO. MED. ASS. J., 92, 348 (1965).

772

23.

S T E R O I D S

In Table I . OOC stands f o r l l - d e o x y c o r t i c o s t e r o n e ; B f o r corticosterone; S for ll-deoxycortisoll F for cortisol; PROG. f o r progesterone; and DOC-SO~- e t c . f o r t h e i r respective sulfates. C21-OAc stands f o r C21 a c e t a t e .

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