Lack of correlation between steroid sulfatase activities and lipid content in uterus and liver microsomes of guinea pigs

LACK OF CORRELATION BET~EN STEROID S~FATASE ACTMTIES AND LIPID CONTENT IN UTERUS AND LIVER MICROSOMES OF GUINEA PIGS S. Roblin,

M. Nicollier,*

3.P.

R&my-Martin,and

C.L.

Adessi

Unite de Recherche de Biochimie Hormonale et des Regulations INSERMU 198, Route de Dole, 25000 Besancon, France Received July 29, 1986 Revised June 12, 1987 ABSTRACT Lipid content and steroid sulfatase activities were determined in liver and uterus microsomes of non-pregnant guinea pigs. The results were compared with values obtained in pregnant and cortisol-treated animals. Steroid sulfatase activities were always higher in pregnant animals, and we supposed that the increase in circulating cortisol in pregnant guinea pigs before parturition has an influence on the membranebound sulfdtdse activities. Sulfatase activities were identical in cortisol-treated and untreated non-pregnant females, although cortisol induced changes in microsomal lipid composition. These results lead us to three conclusions : in intact female guinea pigs, cortisol induces varidtions in the lipid content of uterus and liver microsomes, especially in the cholesteryl sulfate to phospholipid ratios; the variations of the lipid composition in pregnant animals do not appear to be cortisol-dependent; membrane-bound steroid sulfatase activities are not directly influenced by the lipid composition of microsomes. INTRODUCTION In guinea

pig

tissues,

drolase

(EC 3.1.6.113

sulfate

sulfohydrolase

zymes

(1-3).

activities

of

tivity

in the uterus

However,

et al

were increased late

pregnancy,

was increased.

It

in the regulation

hds

or guinea

In human placental

STEROIDS 48 I 5-6

has been reported. pig tissues steroid

sulfate

sulfatase

(3)

[steroid

membrane-bound en-

have reported

in liver only

sulfohy-

that

estrone

been supposed

tract

the

of pregnant sulfatase that

(5)

No such control

sexual

ac-

hormonal

of membrane-bound sulfatases,

(41 and in hamster reproductive

of sulfatases

in human (6)

[estrone

are two related

Moutaouakkil

during

are involved

in rat uterus control

(EC 3.1.6.211

both sulfatases

pigs.

sulfatase

dnd dehydroepiandrosterone

Recently,

guined

factors

estrone

and

hormonal

has been found

(3).

sulfatase

deficiency,

Novem~er~Decem~er

1986

McNaught and

(369-379)

369

370

Roblin et al

France (7) and

McKee et al (8) concluded that the biochemical cause of

the enzyme defect involves a membrane lipid disorder which prevents normal functioning of the enzyme. In sheep brain, Mathew and Ddlasubramanian (9) suggested that estrone sulfatase activity is influenced by the phospholipid composition of membra:es. An attractive hypothesis is to suppose that the increase in microsomal sulfatase activities in pregnant guinea pig tissues is related to the variations of the lipid composition of microsomes. Among the factors inducing changes in the membrane lipids, it hds been shown thdt corticosteroids affect the lipid composition of membrdne and regulate or modulate the activity of the lipid-requiring enzyme systems (10). The present study was designed to investigate the possible involvement of membrdne lipids in the microsomdl sulfdtdse activities. The lipid content and the sulfatase activities of liver dnd of uterus microsomes have been determined in non-pregnant guinea pigs: cortisol-tredted and untreated animals, and the results obtdined were compared with those obtained in pregnant animals. MATERIALS

AND

METHODS

Estrone-3-sulfdte (El-S), dehydroepiandrosterone-3-sulfate (DHEA-S) (sodium sdlt), cortisol,dnd standard lipids were obtained from Sigma Chemical Co (St.Louis, MO, USA). For thin-layer chromatography (TLC), pre-coated silica gel G 60 plates (20 x 20 cm) were obtained from Merck (Darmstddt, FRG). All other chemicals were of anal tical grade. [6,7-‘H]Estrone sulfate (ammonium salt) (53 Ci/mmoll, [7-'H4j dehydroepidndrosterone sulfate (ammonium salt) (24 Ci/mmoll, [1,2-'H]cholesterol (53 Ci/ mmoll, [4- 14CIdehydroepidndrosterone (57.8 mCi/mmol) were purchased from New England Nuclear Corp. (Boston, MA, USA). [4-'4C]Estrone (52 mCi/ 14C]choline dipalmitoyl (58 mCi/mmol) mmoll, .?o-phosphatidyl[N-methylwere purchased from Amersham International (England); [1,2-3H]cholesteryl sulfate (12 Ci/mmol) was synthesized according to Roberts et al (11). Prior to their use, labeled steroids were purified and their purity was checked ds described previously (12). Liquid scintillation spectrometry wds accomplished using 15 ml of scintillation fluid (PiCO-flUOr 15, Packard Instrument Co, Rungis, France) in a Packard Tri-Carb model 460 C liquid scintillation spectrometer with a counting efficiency of 60 % for 'H and 80 % for l“C (double isotope counting with external standardization).

37 1

STEROID SULFATASE AND LIPID CONTENT

Animals ---.

and

treatment

Guined

piqs

Centre

de

in

breeding

our

ddy

dfter

with

gestdtion). ceived

wds

0.9 of

Uteri

%

all

liver

dtus.

in

10 %

Uteri

hdd

dnd

first

were

The

fed

animals

mature

fe-

(60-65

ddys of dnimdls re-

were

then

killed.

ethdnol.

dt

12!500

dt

105,000

with the

50 mM

sdme

1,000

20

min

60

were

w/v)

and

These

buffer

steps

were

in After

minced

with

d Potter-Elvehjem d Virtis

10 min.

pH

dppdr-

Superndtdnts

resulting

superndtdnts

microsomdl

KC1

times

buffer).

finely with

the

The

(TS

with

x g for

min.

mM

severdl

4“C

homogenized

dt

Tris-HCl-150

conditions.

dt

tissues

previously

x g for

wdshed

7.6

: 10,

(I

x g for

dnd pH

both

centrifuged

centrifuged

Lipid

10 ddys

the

mdted

controlled

from

ferndies. Tredted

for

the

They

acid.

femdles

excised

buffer

been

were

centrifuged

under

from

12 D.

:

ascorbic obtdined

buffer

mdteridl, in TS

were fuged

L

were

under

were

(v/v)

rapidly

were

twice

ddy

ddted

pregndnt

mdture per

were

extrdneous

Homogendtes

washed

Tissues

opening),

M sdcchdrose

homogenized

homogenizer.

12

with

from

Femdles

mdintdined

conditions

(SC)

saline

wds

were

enriched

purchased

microsomes --

dnd

scissors,

food

were

Frdnce).

length

Animdls

lighting

vaginal

Tris-HCl-0.25

removing

gestdtion

(1).

cortisol

variety

Montmkdy,

decdpitdtion.

dfter

of

&Grdtion

50 mM

dlbino

dnd cortisol-tredted

2 mg

Vehicle

dnd

by

(6 ddys

dnd

opening standard

sacrificed

males

Hartley

(Cobldnbel,

(20°C,

libitum

were

the

center,

vdgindl

temperature dd

of

Zootechnie

pellets

7.6

and

performed

were

centri-

dt

4'C.

determindtion Lipids

Folch

et

were

dl

(13).

lesterol,dnd

extrdcted

from

Before

extrdction

['H]cholesteryl

covery medsurements. KCI wds dried under

the

microsomdi

pellets

dccording

['4C]phosphdtidylcholine,

sulfdte

were

added

to

the

to

['H]cho-

pellets

for

extract wdshed with in chloroform-methdnol

The chloroform-methdnol NZ gds dnd resuspended

re-

0.1 M (2 :

1,

v/v). The treated to

the

tion

extract by

method

of

was

sepdrdted

perchioric of

Bdrtlett

phospholipids

sulfate.

the

were

three

total

(14).

dnd

Phospholipids

into

dnd

acid,

The

fractions.

phosphorus

second

third

one

Sepdrdted

for

The

wds

one

wds

used

cholesterol

then

They

v/v/v/v).

were

individudlly,dnd v/v). ing

Edch

eludte

wds

Cholesterol dnd to the method of

eluted with

with

visudlized

eluted

with tdken

cholesteryl Nieminen et

M HCL.

The

reacting

with

iodine

phosphorus

for

the

cholesteryl

isold-

:

: 25 : 4, v/v/ 30 : 30 : 6,

vdpors, (10

scrdped

: 10

: 1,

with

dn=

tubuldr

v/

determindtion.

sulfate were sepdrdted by TLC accordal (15). After TLC, cholesterol wds dnd cholesteryl sulfdte (2 : 1, v/v)

eludtes

were

dried

under

N2

gas.

Cholesterol ds trimethylsilyl ether (BSTFA-TMCS, 60 : 20, v/v: dt 60°C) wds qudntified by gds liquid chromdtogrdphy (GLC), with d Frdnce) gds chromdtogrdph equipped Cirdel serie D (Girdel, Suresnes, SE 52. 260°C.

WdS

thin-ldyer

(65 (60

chloroform-methdnol-wdter for

chloroform-methanol

methdnol-1

by

one

according

dnd

by two-dimensiondl

(TLC) using chloroform-methanol-wdter tetrdhydrofurdn-methyldl-methdnol-2 M NH40H

chromdtogrdphy v),

first

medsured

glass-capillary

column

(0.34

The cdrrier gas wds helium (3 mL/min). Trimethylsilyl epicoprostdnol wds used

mm

x 26

m)

codted

The oven temperature ds internal standard.

1 h

with wds

179.3 i 95.0c

n=7

4.63 i-2.62

6.74 + 2.42

Phosphdtidylcholine**

Phosphdtidylserine**

Phosphatidylinositol**

0.003 t 0.001

Cholesteryl sulfate +++ Phospholipids NS

NS

0.004

NS

0.040

0.001

NS

0.024

0.005

(Iv!IIl

a

in

gUined

pig

UterUS

b

0.001

0.001

NS

0.010

0.050

NS

NS

0.020

0.001

0.018

0.001

0.010

0.010

24.85 0.750

NS 0.010

0.010

32.25 3.85

0.020

21.55

10.41

NS

NSd

P" (IVSIII)

21.6

179.5c

Student-Fisher t test. dMean + SDM.

0.003 ? 0.001

0.410 + 0.070

23.55 + 3.79

5.37 + 2.71

8.63 + 4.21

29.88 ? 3.80

19.52 ? 3.50

38.0 + 11.0

571.5 + 86.6c

n=6

lII:Cortisoltreated females (2 mglday for 10 days) Pool

miC!I-OSOIWS

1I:Pregndnt females (60-65 days) P" (IIVSIII)

femdle

ZDifference between groups was tested by Mdnn-Whitney U test. NS: P > 0.05. -1 -1 *Sulfatase activities pmol of product.min .mg prot . **% Totdl phospholipid. ***Moldr ratio.

c1.520 t 0.110

Cholesterol xxx Phospholipids

Phosphatidylethdnoldmine** 15.71 i 4.69

18.23 + 2.40

41.90 k 5.26

Sphingomyelin**

18.3 + 14.5

SUlfdtdSe"

dCtiVitieS

1:Mdture females (6 days after vdginal opening1

SUlfdtdSe

DHEA sulfatase*

Estrone

Number of animals

Group of animals

Table 1 : Lipid content and

=I CD nl

g

op

2

w

STEROID SULFATASEAND LIPID CONTENT

~*m*2

0

ozzzo

d

0

0

N

;;*,m.

c,

ii

+i

. ii

I

ii

373

374

Roblin et al

Cholesteryl sulfdte wds hydrolyzed with methdnol-1 M HCl dt 70°C for lh. Free cholesterol wds then extrdcted 5 times with 5 mL of hexdne dnd qudntified by GLC. These determinations were mdde in duplicdte dnd corrected for methodologicdl losses. Lipid cldsses were cdlculdted in molar amounts dnd results are expressed in percentdges for phospholipids, and in moldr ratios to total phospholipids for cholesterol or cholesteryl sulfdte dccording to Rouser (16). Other

dndlytiCd1

procedures

The protein content wds determined dccording to Lowry et dl bovine serum dlbumin ds stdnddrd. Estrone dnd DHEA sulfdtdse were medsured dccording to our method described previously

with ities

(17) dctiv(3).

RESULTS The estrone microsomes femdles in

dnd DHEA sulfdtdse

from (group

Tables

uterus II),dnd

1 and

microsomal

zymes the

were

mdjor

higher

obtdined

either

activity

ndnt

in

(group

wds higher

wds

femdles

the

in

higher

(group

content

I),

111)

of

pregndnt listed

dre

group

or

liver

microsomes

from

of

mature

uterus

guinea

difference

thdn

the

in

in

both

both

in

sdme group. microsomes (grOUp

in femdles.

to

was observed

or

occurred

phosphoof

in

preg-

III).

cortisol-tredted

between

of

A number

phosphdtidylcholine

pregndnt

en-

micro-

percentdge

sulfdte

of

tissues,

modificdtion

cholesteryl

pig

of

found

d higher

femdles

microsomes

females

In

groups

dnd cortisol-tredted

I contdined

cholesterol

of

liver.

thdt

thdn

the

activities

dctivities

mdture

lower

content

in

or

No significdnt

dnd cortisol-treated

No significant

thdn

higher

tissues

the

sulfdtdse

between

from

the

dnimdls,

microsomes

wds observed

lipid

II)

of

dnimdls.

thdn the

lipid

(group

wd5 dlwdys

of

pregndnt

dnd

ratios

femdle5

dctivity

groups

uterus

microsomes

The content

mdles.

in

from

sphingomyelin

chdnges

sulfdtdse

corresponding

some5

lipid

mdtUre

cortisol-tredted

estrone

fedture

Uterus

of

DHEA sulfdtdse.regdrdless

In

dnimdls.

liver

dnd the

2.

Microsomdl of

dnd

dctivities

pregndnt

(Tdble

1) fe-

dnd cor-

tisol-treated pregnant

females.

STEROID SULFATASEAND LfPiD CONTENT

375

Phosphatidylethanoiamine

in

and cortisol-treated

animdls

sum of phosphatidyicholine tively

constant

be supposed

thdt

the increase

dnd cortisoi-treated

animals (18).

inositoi

wds increased

females.

Cholesterol

uterus

microsomes

content

females

untredted

myelin

were observed

was higher

or pregndnt observed sulfate

females

in pregnant

to phospholipid wds

or pregnant

rdtio

noticed

rdtio

females.

was observed

between pregndnt

intact

ratios

in

than in pregbetween

in

phosphatidyicboline, The most significant

animals, lower

where sphingothdn

in untreated

in phosphdtidylinositol

and untreated increased

in

2) was identical

fern&es.

increase

or

was noticed

for

in cortisol-treated

A significant

to phospholipid

females

(Table

except

It can

phosphdtidyl-

in pregnant

difference

microsomes

between cortisol-treated

pdred to untreated

change

liver

%I.

to phosphoiipid

and phospbdtidylethdnolamine

females.

rela-

in pregnant

femdles,

sulfate

No significdnt

decreased

remained

by the decredse

in cortisof-tredted

dnd pregnant

which wds slightly differences

In cortisol-treated

of

The

to 57.60

wds counterbalanced

when compared with that

ndnt or mature femdles.

The lipid

(53.43

females.

in phosphatidylethdnoiamine

were higher

groups.

than in untreated

of animals

or cholesteryl

the two latter

was higher

and phosph~tidylethanolamine

in the 3 groups

phosphatidylcholine

content

WdS

females.

Cholesteryl

in cortisol-treated

femdles

The lowest

in untredted

value

animals.

dnd cortisol-treated

of

com-

cholesterol

No significant fern&es.

DISCUSSION In guinea fdtdse

confirms

pig uterus

activities

were

our previous

dnd liver

always

results

microsomes,

the highest

concerning

estrone

in pregnant the increase

dnd RHEA sulfemales, in steroid

and this sul-

376

Roblin et a/

fdtdses

in

origin

of

sults

were

(4-6). the

liver this

In

reported

control

d

of

few

ddys

membrdne-bound mdles,

the

in mdture The

gested

before

hypothesis

of

is

dlso

literdture

somes.

The

comparison

phospholipid reldtively

low

is,

Cortisol lipid

in

uterus

induces

of

creased

dnd

dfter

thdt in

present rdt

of in

liver dn

thdt

No

liver

pig

of

d reldtive

incredse In

in rat

both

cortisol

those (10) dnd

ddministrdtion.

found

membrdne-bound have

sug-

in

of

microsomes,

reported

for

of

th?

the

phosphd-

dddition,

sphingomyelin. chdnges in dnd Melby

sphingomyelin effect

rel-

micro-

In

d decredse

The

the

uterus

dlso

significdnt

dnd

fe-

emphdsizes

sphingomyelin

liver

the

dnimdls.

pig

dbunddnce

phosphdtidylethdnoldmine

vivo

guined

microsomes.

ndmely

guined

dctivities

dnd

We

influence

dvdildble

with

femdles

20).

composition.dnd

dre

microsomes

microsomes,

rdtio.

ddtd

liver

might

some-duthors

phosphdtidylcholine

guined

(19,

pregndnt

lipid

results

ddrendlectomy

pregndnt

the

cortisol

composition

cortisol-tredted

phospholipid

reported

(22).

microsomes,

in

tidylethdnoldmine to

of

found

composition

sulfdte

the

percentdge

fdct

under

cortisol-tredted

to in

corticosteroids,

lipid

of

composition

tidylethdnoldmine there

the

between the

dctivities

for

found

in

(21)

identicdl

those

dnd

The

re-

being

enzymes

in

(3).

sulfdtdse

sulfdtdse

cortisol

However,

reldtionship

dmong

of

microsomdl

were

by

membrdne-bound

pdrturition

dctivities.

supported

enzymdtic

of

pigs

conflicting

Corticosteroids

of

thdn

guined

species, of

circuldting

onset

lower

d reldtionship

evdnt

have

the

other

evidence

(3).

dctivities

dnd

microsomdl

of

sulfdtdse sulfdtdse

no

d number

incredse

femdles

sulfdtdses

of

pregndnt

Control

is

hormones

dctivity the

there

of

In

d hormondl

pig

ovdridn

thdt

homogendtes

is unknown.

for

guined

the

supposed pigs

incredse

the

influence

uterus

dnd

of

in

the

phosphdcholesteryl et

dl

(10)

levels

de-

cortisol

on

377

STEROID SULFATASEAND LIPlD CONTENT

the phospholipid fic.

composition in guinea

Moreover,

lipid

composition

ferent

that

induces

(241.

ratios.

sulfdtdse

In guinea

activities

the phospholipid similar

males.

However,

ratios

in uterus

These results uterus

microsomes

cortisol

suifdtdse

at the end of

pregnancy

deficiency,

phospholipids,

particularly

Mathew and Balasubrdmanian

cortisol

to phosphomembrane

on membrdne-bound

changes

microsomes.

are observed

These alterations

in cortisol-treated sulfate

females

in are

mature fe-

to phospholipid

are identical

ds in cortisol-treated in the cholesterol

to those females.

content

level

of

of circulating

(21).

sulfatases

Mc Kee and France microsomes

cholesterol

sulfate

by the increasing

between the lipid

of membrane-bound

sulfatdse-deficient jor

the changes

are not induced

The relationship expression

that

mem-

dnimals.

or cholesteryl

and did not increase

plasma and

of cellular

known to modify

by cortisol

pregnant

dif-

reported

microsomes,

and cholesteryl

of uterus

of

kidney

modifies

and liver

pig the most striking

induced

are quite

hdve dlso

they did not have any effect

microsomes

suggest

In rat (23)

speci-

in the phospho-

changes

Cortisol

are widely

the cholesterol

of mature females

these

membranes. pig uterus

composition

to those

chdnges

microsomes.

in cortisol-treated guinea

to be Species

upon the composition

in cholesterol

However,

In pregnant

very

all

These variations

(25).

induces

and Milkovic

of corticosteroids

an increase

fluidity

in liver

for

appedrs

microsomes.and

Ozegovic

Wds not similar

biosynthesis

lipid

noticed

membranes,

the effect

brdnes

pig cortisol

of uterus

from those

brushborder

of microsomes

content is

of microsomes

not proved.

(26)

levels

phosphatidylcholine.

(91 demonstrated

In human steroid

showed that

have subnormal

that

and the

of

the steroid each of

the ma-

In sheep brain, phospholipase

A could

378

Roblin et a/

completely inactivateestrone sulfatasewithout affecting arylsulfatase C. They concluded that the lipid requirementof the two enzymes for catalytic activity was different.In guinea pig liver and uterus microsomes, the variationsof the lipid content are not correlatedwith the estrone and DHEA sulfatase activities.The independenceof membranebound sulfataseactivitiesto their lipid environmentis supported by the observationsof Van der Loos et al (27) and Epstein and Bonifas (26). who suggest that the X-linked placentalsteroid sulfatase deficiency is due to a strongly decreased amount of steroid sulfatase protein rather than a membrane disorder. Our results lead us to three conclusions : in guinea pig, in vivo, administrationof cortisol to untreated females induces variations in the lipid content of uterus and liver microsomes, especially in the cholesterylsulfate to phospholipidratio; the variations of the lipid compositionof uterus and liver microsomes in pregnant animals do not appear to be cortisol-dependent; membrane-bound estrone and DHEA sulfataseactivitiesare not directly influencedby lipid compositionof microsomes. ACKNOWLEDGMENT

We are grateful to Myriam David for the typing of the manuscript. REFERENCES 1.

2. 3. 4. 5. 6. 7.

Adessi, G.L., Nhuan, T. Q.,and Vingler, P., 3 STEROID BIOCHEM 16, 107 (1982). Freeman, D.S., Saidi, F.,and Hobkirk, R., 3 STEROID BIOCHEM Is, 23 (1983). Moutaouakkil,M., Prost, O., Dahan, N.,and Adessi, G.L., 3 STEROID BIOCHEM 2J, 321 (1984). Utaaker, E. and Stoa, K.F., HORM RES 13, 180 (1980). Legault, Y., Bleau, G., Chapdelaine,A.,and Roberts, K.D., 6IOL REPROD 23, 720 (1980). Prost, O., Turrel, M.O., Dahan, N., Craveur, C.,and Adessi, G.L., CANCER RES ,4, 661 (1984). McNaught, R.W. and France, J.T., 3 STEROID BIOCHEM 1;1,363 (1980).

STEROID SULFATASEAND LIPID CONTENT

8. McKee, 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 70. 21. 22. 23. 24. 25. 26. 27. 28.

J.W.A., Abeysekera, R..and France, J.T., 3 STEROID BIOCHEM 14, 195 (1981). 3 NEUROCHEM 2, 1205 (1982). Mdthew, 3. dnd Bdldsubrdmdnidn, A.S., Melby, J.M., Wennhold, A.R.. dnd Nelson, D.H., ENDOCRINOLOGY 109, 920 (1981). Roberts, K.D., Bdndi, L., Calvin, H.I., Drucker, W.D..dnd Liebermdn, S ., BIOCHEMISTRY 3, 1983 (1964). Prost, 0. dnd Adessi, G.L., 3 CLIN ENDOCRINOL METAB 56, 653 (1983). Folch, 3., Lees, M..dnd Sloane-Stanley, G.H., 3 BIOL CHEM 226, 497 (1957). 3 BIOL CHEM E, 466 (1959). Bdrtlett, G.R., Nieminen, E., Leikold, E., Koljonem, M., Khstdld, U..dnd Mustdkdllio, K.K., ACTA DERMATOL 47, 327 (1967). Rouser, G., in : Membrdne Fluidity in Biology (Aloia, R.C., Ed) New York, Vol I (1983) pp 235-289. Acddemic Press, Fdrr, A.L..dnd Rdnddll, R.S., 3 Lowry, O-H., Rosebrough, N-3., BIOL CHEM E, 265 (1951). ENDOCRINOLOGY 118, 244 (1986). Moulton, B.C. dnd Koening, B.B., Litwack, G., in : Topics in Medicindl Chemistry (Rdbinowitr, 3.L. New York, Vol I (1967) pp 3-36. dnd Meyerson, R.M., Eds), Wiley, Cdstro, J.A., Green, F.E., Gigon, P., Sasame, H..dnd Gillette, 3.R., BIOCHEM PHARMACOL 19, 2461 (1970). Jones, C.T., ENDOCRINOLOGY 95, 1129 (1974). Nelson, D.M., ENDOCR REV I, 180 (1980). Oregovic, B. dnd Milkovic, S., PERIOD BIOL 9, 355 (1983). Picdrd, F., 3 STEROID BIOCHEM 12, 253 Homo, F.,dnd Duvdl, D., (1980). Le Grimelec, C., Ddignedult, A., Bledu, G.,dnd Roberts, K.D., LIPIDS Is, 474 (1984). McKee, 3 STEROID BIOCHEM !.& 309 (1983). 3.W.A. dnd Frdnce, J.T., Vdn der Loos, C.M., Vdn Bredd, A.3., Van der Berg, F.M..dnd gobsis, A.C., 3 STEROID BIOCHEM 2, 1743 (1983). Epstein, E.H. dnd Bonifds, 3.M., HUM GENET 2, 201 (1985).

APPENDIX Abbrevidtions Bis (trimethylsilyl) trifluoroacetamide Trimethylchlorosildne (TMCS) Thin-ldyer chromatogrdphy (TLC) Gds liquid chromatography (GLC)

(BSTFA)

NOTE *To

379

whom

correspondence

should

be

sent.