Monosulfates of 16-oxygenated ketonic C19 steroids in adult human urine

235

MONOSULFATES

OF

STEROIDS

Cl9

0. Department

Received

of Medical

June 5,

16”OXYGENATED IN ADULT

Janne

and

Chemistry,

KETONIC

HUMAN

R.

URINE

Vihko

University

of Helsinki,

Finland

1969

ABSTRACT A monosulfate fraction of steroids in human urine was obtained by chromatography on Sephadex LH-20. After solvolysis, fractionation on silicic acid and thin-layer chromatography, the steroids were subjected to gas-liquid chromatographic and gas The following chromatographic-mass spectrometric analysis. 16-hydroxylated ketonic C 1 9 steroids were identified in the monofemale and male urine: 16a-hydroxydesulfate fraction of normal hydroepiandrosterone, 16a-hydroxyandrosterone, 16a-hydroxyepi16a-hydroxyetiocholanolone, 3a, 16a-dihydroxyandrostandrosterone, -5 -en-l 7-one, and 16P-hydroxydehydroepiandrosterone. In addition, 3p, 17P-dihydroxyandrost-5-en-16-one was shown to be present in the same fraction of urine. Some quantitative values of the urinary excretion of these compounds are given. INTRODUCTION It serve

is

well

as

precursors

(summarized

documented

in

identified

in

cord

plasma

blood

present shown

in

ponent

16a-hydroxylated

estriol

pregnancy

biosynthesis

plasma

(2, 3),

(3) and in infant male

urine

infant

several

female,

steroids

were

-16”one

has

and

female

The reflect maternal

in

urine

(6).

urine

identified been

(5).

steroids the

and

on

and

in the urine in normal male

excreted

combined

tissues

pregnancy

effects their

(11) in

a

is has

normal

male

in also been

normal

administration

nonketonic

comof

and

a

16a-hydroxylated

(8). 3p, 17P-dihydroxyandrost-5-eninfant

urine

(9),

amniotic

fluid

(4)

urine.

maternal of

it

been

(4),

steroid

recently is

a

fluid

This

After to

ketonic

identified

(10)

steroids

has

amniotic

Quite

(7).

16a-hydroxydehydroepiandrosterone

in

during

16P-hydroxydehydroepiandrosterone

of l-3-day-old

pregnant

Cl9

16a-Hydroxydehydroepiandrosterone

1).

normal

that

in

that

the

precursors.

urine

fetus, The

during

the

pregnancy

placenta

immediate

and neutral

the

14:3

STEROIDS

230 of

precursors tized

to

a

transported that

by

great to

by

vestigations gas

different

normal

about

paper

and gas

and males.

Urine was of age.

but

the

state

in

describes

Thus

maternal

of the fetus As

ketonic

C

A preliminary

published

19

fetus,

obviously

(8, 12).

steroids the

by

a the

step

aromapartly

it

was

urine,

felt more

and placenta in

these

in-

identification

chromatography-mass

by

spectrometry

steroid

sulfates

report

of part

of

in urine of this

of

work

(13).

MATERIAL years

placenta

determinations.

present

been

produced

circulation

16-oxygenated

females

already

the

neutral

be gained

estrogen the

in

are

maternal these

chromatography

seven

has

the

could mere

estrogens extent

analyzing

information than

the

AND

METHODS

obtained from healthy females The samples were stored at

and males, 20-30 -20° until analyzed.

All solvents were of reagent grade and were redistilled before use. Reference steroids were obtained from Prof. W. Klyne (Steroid Reference Collection, London, England) and Prof. S.Solomon (Royal Victoria Hospital, Montreal, Canada), or purchased from Ikapharm (Ramat-Gan, Israel). 16a-Hydroxyepiandrosterone was obtained by hydrogenation of 16a-hydroxydehydroepiandrosterone, using palladium as catalyst. Ref. 25 gives the trivial names of the steroids used in the present study and the corresponding systematic names. Procedure. The monosulfate fraction of the steroids in urine was obtained as described in detail earlier (14). In outline,the procedure is as follows: 1) Evaporation of the urine (1 O-20 ml) in vacua at 39OC. 2) Chromatography of the urinary extract on Sephadex-G-mine, using the solvent system chloroform/methanol/water (60/30/5 by vol.). 3) Isolation of the monosulfate fraction of the steroid conjugates by chromatography on Sephadex LH-20. 4) Solvolysis in ethyl acetate acidified with sulfuric acid, The liberated steroids were fractionated on a 3-g silicic acid column (15). The fraction eluted with 20 ml of ethyl acetate contained 16”oxygenated ketonic Cl9 steroids, some nonketonic 16-hydroxylated Cl9

steroids

and polyhydroxylated

C21 steroids.

This

further purified by thin -layer chromatography. Thin-layer chromatography (TLC) of steroids was

fraction

carried

was

out using

20 x 20 cm precoated abrasion-resistant Silica gel GF254 _ layers (Merck AG, No. 5715, 0.25 mm). The solvent system chloroform/absolute ethanol (90/10 by vol.) was used and ascending chromatography was carried out twit e. The zone containing 16-oxygenated ketonic Cl9 steroids was scraped off. The material eluted with methanol was further subjected to gas-chromatographic and gas chromatographic-mass spectrometric analyses. Gas-liquid chromatography (GLC) of s t eroids was performed on QF-1, SE-3 cphases, trimethylsilyl (TMS) and usin 0-methyloxime-trimethylsilyl (MO-TMS 9 ether derivatives as described earlier (16-18).

STEROIDS

Sept. 1969

237

Gas chromatography-mass spectrometry (GC-MS) of TMS and MO-TMS derivatives was performed on the LKB Model 9000 Gas Chromatograph-Mass Spectrometer (LKB-Produkter AB, Stockholm-Bromma, Sweden), using QF-1 and SE-30 liquid phases during the GLC (16,17). The compound was considered to be identified when the relative retention times as TMS and MO-TMS ethers and the mass spectra of these derivatives were the same as those of the appropriate reference steroid. In addition, the steroids were reduced with sodium borohydride in ethanol overnight and the derivatives so formed analyzed as TMS ethers by GLC and GC-MS. Comparisons of the relative retention times and mass spectra with those of the corresponding derivatives of reference steroids gave further support to the identifications.

RESULTS Fig.

1 shows

of urinary Cl9

steroids

steroids

from

analyzed

shows

analyses

SE-30

columns.

(numbered

the gas

chromatographic the fraction

on QF-1,

The

I-VII)

relative

in this

of the corresponding

of these

retention are

of the TMS

16-oxygenated

and XE-60

ethers

fraction

reference

containing

SE-30

of the MO-TMS

analyses

times given

compounds

columns.

steroids (RRT)

ethers ketonic

Fig.

on QF-1

2 and

of the compounds

in Table

1 and the GLC data

in Table

2.

retention times (relative to 5a-cholestane) of steroids Table 1. Relative identified in the fraction of 16-oxygenated ketonic C steroids. Cholestane time : QF-1 10-l 1 min., and XE-60 14-15 SE-30 28-29 r%n. min. For conditions in GLC see Fig. 1. For identification of the compounds see Table 2 and text. Compound

QF-1 TMS

SE-30

MO-TMS

TMS

XE-60

MO-TMS

TMS

MO-TMS

1.02

0.53+

0.60+

0.59+

0.93

0.59+

II

1.13+

0.58

0.64

0.64

0.99+

0.62

III

1.13+

0.53+

0.60+

0.59+

0.99+

0.59+

IV

1.43

0.74

0.84

0.88

1.33

0.87+

1.59

0.80++

0.87++

0.90++

1.41

0.87+

VI

1.65

0.98

0.92+

1.03

1.49

1.08

VII

1.80

1.04

0.92+

1.11

1.60

1.20

I

V

t tt

Mixture In

the

of latter

compounds. part

of the

peak.

238

STEROIDS

14:3

IV

OF-I. TMS

II n

VII

I

I

10

VII VI

n

IV

SE-~~.TMS

11

XE-GO.TMS

VII

V VI

i -_: 210

Figure

110

WIUTES

1. Gas chromatographic analyses of the TMS ethers of 16-oxygenated steroids in human urine. Columns and conditions: 3 % QF-1, 215O C, 2.2 % SE-30, 225OC, 3 % XE-60, 225OC. The analyses on QF-1 and XE-60 columns are from urine of a normal female (aged 20 years), and the analysis on SE-30 is from a urine pool of normal males. Peak identifications : see text.

Table

2. 1.

1.04

1.66 1.80

16P-hydroxydehydroepiandrosterone

30,17P-dihydroxyandrost-

here.

This

.

was

The

17-ketonic

columns

x, All

also

relative

the

main

retention peak

time

MO-TMS

0.97

1.60

16a-hydroxyepiandrosterone

steroids

0.80

1.43

16a-hydroxydehydroepiandrosterone

-5-en-16-one

0.74

1.15

of

of

each

the

derivatives

0.54

0.58

16a-hydroxyetiocholanolone

TMSX)

in

of

of

each

peaks

1.11

1.04

0.90

0.87

0.58

0.64

0.59

QF-1

is

and

1.64

1.48

1.40

1.31

1.03

1.00

0.93

TMS

listed

XE-60

1.20

1.08

0.89

0.86

0.60

0.63

0.59

MO-TMSX)

XE-60

1.

steroids. Fig.

compound

on

see

reference GLC

MO-TMS

SE-30

two peak steroid.

first

gave

0.94

0.92

0.87

0.84

0.61

0.65

0.60

TMS

conditions

5a-cholestane)

For

to

0.53

MO-

QF-1

Table

(relative

1.12

as

in

TMS

times

indicated

16a-hydroxyandrosterone

as

retention

1.02

s

3a, lba-dihydroxyandrost-5-en-17-one

time

Relative

Compound

Cholestane

240

STEROIDS

14:3

I”

\

L 111

1

, I

I

30

20

Figure

2.

Gas

I

YINUTES

10

10

chromatographic

analyses

of

the

MO-TMS

of 16-oxygenated steroids in human urine. Columns 3 ‘$I QF-1, 215OC, 2.2 % SE-30, 225OC. The analysis column: female urine, on the SE-30 column: male Peak identifications: see text. Fig. 1). Compound was

I.

eluted

the

liquid

identical

This

compound,

as

faster

than

any

phases

used

during

with

the

other

reduction

pound

I

had

duction

product

(QF-1 as

RRT

0.70,

the

TMS

of

sodium

reference

0.89).

ether

showed

175,

m/e

448

196,

199,

(Fig.

3).

214

with

derivative,

fraction had

of

The a

mass

reference

compound

I

3a, 16a-

XE-60

columns.

the

TMS

ether

of

the

TMS

ether

spectrum

304

values

and

of

typical

intense

peak),

all

SE-30

fragmentation

(base

on

RRT

of

3a, 16a-dihydroxyandrost-5

i.e. --

Thus,

this

steroid

borohydride,

-dihydroxyandrost-5-en-17-ones, 129,

QF-1,

identical

~~-30

The

and conditions: on the QF-1 urine (see

MO-TMS

in

derivatives on

values

and

steroid

corresponding

with

TMS

GLC.

-dihydroxyandrost-5-en-17-one After

the

ethers

was

a

re-

7-one

compound of

I

3,16at m/e

molecular

identified

the

-en-l

fragments

and

com-

ion

at

3a, 16a-

as

-dihydroxyandrost-5-en-17-one. Compound

II.

spectrum 450,

base

117,

196,

had 4,

a

TMS

(Fig.

4,

lower

peak

at

201

mass

upper

mass

The

ether panel)

m/e

216

and 306.

The

spectrum

with

panel)

spectrometric

and

of

the

this

with

and

base

fragmentation

the

other

MO-TMS the

compound molecular intense

ions

derivative

molecular peak

gave

at

of

ion m/e

patterns

at

448 of

the

a

mass

ion

at

at

m/e

m/e 106,

compound m/e

(M-31). two

479

II (Fig.

The derivatives

241

STEROIDS

Sept. 1969 I

COMPOUND

214

I

TMS

304

‘75

II

199 196 \

,L

I

100

200

Figure

The

3.

300

we

mass

spectrum

of

400

compound

I

as

the

COMPOUND

TMS

ether.

II

MO -TMS

16

COMPOUND

II

TMS

306

1,

m/e

Figure (below) of

The

4. and

mass

spectra

MO-TMS

of

values

of

were

hydride

reduction,

values -trio1

the

identical,

same

tri-TMS

trum

typical

bons

3,

compound

16

of

as

II

as

II

as

those

as

the

with

reference

seen

in

Tables

II,

as

the

of

(QF-1

(19).

identical of

reference

0.72,

androstanetriols 17

compound

and

compound

ether

and II

were

compound

sterone

436450 I

I 400

TMS

derivatives.

16a-hydroxyandrosterone

RRT

345

I 300

These

having

16a-hydroxyandrosterone.

II. The

16a-hydroxyandro1

TMS

and

2.

ether,

After had

0.92)

and

hydroxyl

confirm

the

a

boroRRT

5a-androstane-3a,

SE-30

data

compound

16a, mass

groups

17p-

spec-

at car-

identification

of

14:3

STEROIDS

242

III.

Compound rated

from

the

Figs.

peak

CC-MS a

TMS

observed

steroid

with

and

at

stane-3a,

16a,

compound

the

seen

an

I in

not

its

as

identical

RRT

as

in

seen

III

Compound

IV.

in

of

to

and

identified

fraction

were

of

these

GLC

basis

Its

the

derivatives The

RRT

same

had

mass

spectra

the

basis

of

had

base

peak

106,

196,

steroid

gave

the

pattern

of

and

the

as

GLC

and

TMS

Tables

1

and

at 216

ether of the

III

these

and

re-

had

results

as

TMS

of

the

comand

correspondThe

ions

at

mass

m/e

compound

IV

ethers

448 and

are

data

TMS

reference 2.

the ion

m/e

117

and

306.

ion

at

TMS at

and

16a-

presented

derivative

as

of

After

ether GLC,

of

compound

V

of this

V

was

typical

steroids 3p, 16a-dihydroxy-

had

16a-hydroxyepiandrosterone, borohydride

were

fragmentation

compound

reference

in Fig.

ions

ether

The

that

seen

intense

MO-TMS 479.

was

revealed

450,

other

The

compound

ether

m/e

and

m/e

this

3, 16-dihydroxyandrostan-17”one

On

this

of

molecular

molecular

with

with

a

201,

-5a-androstan-17-one.

in

of

compound

values

MO-TMS

GC-MS

analysis

was

derivatives

identical

identical

spectra

5.

compound

such

the

5p-andro-

major

of

CC-MS

of

had

the

molecular

V.

m/e

III

boro-

II

those

Compound

at

After

was

as

16a-hydroxydehydroepiandrosterone.

The

the

compound

of

steroid

as

6.

as

reference

of

identified

this

450

con-

16a-hydroxyetiocholanolone,

the

this

-hydroxydehydroepiandrosterone

On

peak

16a-hydroxydehydroepiandrosterone.

respectively.

Fig.

m/e

during

16a-hydroxyetiocholanolone.

studied.

derivatives

of

as

this

phases

column

and

compound

those

reference On

SE-30

mass

sepa-

liquid

ether.

The

On

2.

the

part

as

ether.

Quantitatively,

derivatives

spectra

1

was

the

MO-TMS

with

Tables

compound

pound

values

the

at

column

similar

on

MO-TMS

derivative

tri-TMS

on

partly

symmetric

ion

the

only

II

latter

16a-hydroxyetiocholanolone.

ference

in

is

SE-30

very

is

that

molecular

TMS

on

were

is

479

17/3-trio1

III

it

that

the

compound compound

compound

m/e

(0.83)

this

and/or 2

was

RRT

477,

and

reduction

same

ing

1

by

ether

hydride

I

formed it

tained

GLC

compound

In

used.

During

reduction,

RRT

values as

compound

seen V

I

243

STEROIDS

Sept. 1969

I

46

266

COMPOUND

I

IV

MO-TMS

3.56 462

161x-HYDROXYDEHYDROEPIANDROSTERONE

100

200

MO-TMS

400

300 m/e

Figure

The

5.

reference derivatives.

mass

spectra

of

compound

IV

16a-hydroxydehydroepiandrosterone

7

(above)

as

216

and

of

the

MO-TMS

COMPOUND

V TMS

I

196

I

306

\ 201 IIII

k

450

I,1

I

,,J

,I

200

Figure had

the

trio1

same

16

and

of 17

identification VI.

compound

was

The ion

the

at

(19).

fraction

mass m/e

values

as

(QF-1

The

As

II

I 400

both

reference

eluted

later

studied

and

on

of

with

all

other

ions

and the

the

compound

the

TMS

hydroxyl

and

a

16a, mass

groups

analyses

ether. 17p-

spec-

at carbons

confirm

the

16a-hydroxyepiandrosterone.

TMS than

as

1.22)

GC-MS

as

V

! !

5a-androstane-3P,

SE-30

and

V

the

compound

1.03,

GLC

compound

spectrum 448

of

androstanetriols

of

Compound

in

RRT

spectrum

ether

typical

I

300

mass

tri-TMS

trum 3,

The

6.

1

I,

435 1

MO-TMS other

liquid VI

derivatives

16-hydroxylated phases

TMS

characteristic

used

ether

had of

this

this steroids

during a

GLC.

molecular derivative

244

14:3

STEROIDS

of

3,16-dihydroxyandrost-5

sterone

and

the

TMS

tra

of

ions

cal

m/e

VI

with

di-TMS

The

7-ones.

have

only

marked

are

seen

in

129,

and

304

(7).

as

the

that

of

214 TMS

ether

the

(Fig.

is

reference The

7).

16P-Hydroxydehydroepiandro-

very

steroids

ether

I

16a-isomer

ethers.

these

at

pound

its

-en-l

similar

mass

differences the

in

relative

The

as

mass

spec-

the

intensities

mass

presented

spectra

of

spectrum

in

Fig.

7

of

and

the com-

is identi-

16/3-hydroxydehydroepiandrosterone MO-TMS

ether

of

this

compound

had I

I

214

I I

129

COMPOUND

I

VI

TMS

304 I

I

I

I

I

lb

IQ’--HYDROXYOEHYOROEPIANDROSTERONE

m/e The mass spectra of compound Figure 7. ence 16P-hydroxydehydroepiandrosterone as a

molecular

had

ion

RRT

at

values

m/e

identical

hydroepiandrosterone 2).

Thus

with

on

compound

VI

During

477.

all was

GLC

those

the

VI (above) the TMS

of

liquid

analyses

reference phases

identified

as

and of ethers.

refer-

compound

VI

16P-hydroxyde-

used

(Tables

1 and

16P-hydroxydehydroepi-

androsterone. Compound the

fraction

gave

mass

steroids pound with

VII.

studied spectra

during of

In

the se

compound

addition also

to

3, 16-hydroxylated

contains

some

characteristic

GC-MS (compound VI during

of

analysis VII) GLC

on

compounds

dihydroxy

(Figs. was

other

17-ketosteroids

1 and

eluted the

monoketo 2).

The

slightly

columns

used.

which C

main

after The

19 com-

or along mass

245

STEROIDS

Sept. 1969

COMPOUND

VII

TMS

433

Figure

8.

The

mass

the

TMS

spectrum

of

compound

VII

as

the

I

TMS

ether. spectra

of

compound

VII

derivatives

were

of

values

RRT

ether

(Fig.

identical

with

and

MO-TMS

those

of

derivative

the

of

the

derivatives

of

VII

was

therefore

Further,

compound

were

of

corresponding

3p, 17P-dihydroxyandrost-5-en-16”one.

-dihydroxyandrost-5-en-16-one Compound

8)

VII

identical

and

(Tables

the

3S, 17pm 1

and

identified

as

3p, 17P-dihydroxy-

moment,

no

recovery

2).

androst-5-en-16-one. Quantitations. with

the

have

been

At

sulfate

the

present

conjugates

carried

of

The

out.

16”oxygenated recovery

hydroxydehydroepiandrosterone, and

and

was

procedure, the

carried

urinary

Table

3

the

per

excretion

are

-en-l

through

70-80

of

uncorrected

ketonic

C

unconjugated

steroids 19 16a-

16P-hydroxydehydroepiandrosterone

3p, 17P-dihydroxyandrost-5

medium

of

experiments

cent.

the

6-one,

The

steroids

for

added

different

to

steps

quantitative analyzed

methodological

the

in

solvolytic

the

values given

of

in

losses.

DISCUSSION A human After silicic

monosulfate urine

was

solvolysis acid

and

steroids

was

steroids

in

performed

fraction obtained

using

by

neutral

liberated

steroids

the

fraction

containing

purified

fraction GLC

as and

steroid

conjugates

chromatography

the

further this

of

on

were

Sephadex

fractionated

16”oxygenated

on

TLC.

The

TMS

and

MO-TMS

GC-MS

analyses

in

on ketonic

identification

on

derivatives the

LH-20.

QF-1,

of

Cl9 the was

SE-30

ml.

of

the

the

36, 17/3-dihydroxyandrost-5-en-16-one

136

52

16P-hydroxydehydroepi-

androsterone

38

555

6

124

12

Mean

Females 3

pg

of

16a-hydroxyepiandrosterone

16a_hydroxydehydroepiandrosterone

16a-hydroxyetiocholanolone

16a-hydroxyandrosterone

as

excretion

expressed

Urinary

Steroid

pg/lOO

are

3.

3a, 16a-dihydroxyandrost-5-en-17-one

as

values

Table

5 -

86)

56)

” 730)

7)

( 60 - 182)

(19-

(

(211

(<5-

15)

- 220)

8 -

( 27

(

years,

steroid/24

(Range)

(20-30 subjects)

free

monosulfates

of case

241

73

36

573

12

80

9

-

(138

40)

149)

16)

116) - 360)

-

65)

- 1060) 15 ( 38

(

(420

(<5”

( 28 -

(<5

(Range)

the

Mean

in

(20-30 years, subjects)

and

ketonic

Males 5

hrs

16-oxygenated of

31.5

10.3

3.7

92.0

2.6

7.5

1.6

subjects, LO-30 years)

urine

urine

(fronri 8

male

The

male

pooled

steroids.

Pooled

Cl9

and

XE-60

with as

liquid

sodium the

ethers

question procedure

dihydroxy

monoketo

Sephadex was

The

17.

observed

tion

ditions

is

crucial

(20,21)

nor al.

of

free

sterone,

place

reference

have

medium.

any

was

GLC

reported

To

of

same

and

(70-80 were

solvolysis.

per

TLC,

the

cent)

and

the

same

During

these

as

after of

the

the

certain

con-

into

3p, 17p-

artifact

forma-

procedure,

a mixture

3@, 17P-dihydroxyin

each

solvolytic silicic

steroid

relative

no

the

solvolysis,

original

procedures

with

16a-hydroxydehydroepiandro-

recovery that

no

incubation

that

in

that

and

to

carbons

5a/5P-isomeriza-

under

analytical

consisting

revealed

of

converted

check

present

at

of

3a/3p-isomerization

that is

subjected

analysis

no

the

analysis

conjugates

sign

16P-hydroxydehydroepiandrosterone

chromatography

steroids

the

steroids

androst-5-en-16-one

the

there

the

during

functions

solvolysis

(7).

during

in

steroid

16P-hydroxydehydroepiandrosterone

takes

reduced analyzed

occurs

oxygen for

During

-dihydroxyandrost-5-en-16-one tion

with

was

were

formed

formation

conditions

mild.

so

importance

Cl 9 steroids

are

steroids

GC-MS.

artifact

of

et --

the

derivatives

and

whether

Shackleton

(21).

the

GLC

isolation

LH-20

addition,

and

by of

analytical

and

In

phases.

borohydride

TMS

The

16

247

STEROIDS

Sept. 1969

amounts

mixture

formation

acid

was of

about the

subjected of

to

artifacts

was

observed. 16a-Hydroxydehydroepiandrosterone from

the

urine

of

-dihydroxyandrost-5 and

male

steroids

(11)

normal -en-l

urine.

identified,

epiandrosterone,

male

not urine.

identified

and

been

(12,6)

infant

reported

urine

as

identified

in

female

(10)

ketonic

Cl9

16a-hydroxy-

3a, 16a_dihydroxy-

constituents

(7).

After

a

normal

(8, 12)

Solomon

in

3p, 17p-

16@-hydroxydehydroepiandrosterone,

YoungLai

and

isolated

and

160oxygenated

of

normal

16p-Hydroxydehydroepiandrosterone in

been

identified

16a-hydroxyetiocholanolone,

to

sterone,

males

been

five

already

16a-hydroxyandrosterone,

dehydroepiandrosterone

steroids

and

has

other

namely

androst-5-en-17-one hitherto

females

6-one

The

has

the

16a-hydroxyetiocholanolone

administration male

isolated

present

‘was

and

and

study, and

and

16a-hydroxy-

pregnant

identified namely

female recently

of a

have

three

female, of

the

16a-hydroxyandro-

3a, 16a-dihydroxyandrost-5

-

14:3

STEROIDS

248 -en-l

7-one.

Their

-5”en-17-one

is

studies the

indicate

most

that

abundant

3a, 16a-dihydroxyandrost-

metabolite

of

16a-hydroxy-

dehydroepiandrosterone,

and is possibly formed as a result of 4 by the 16”hydroxyl group of the enzyme A lisomerase 3 injection of [7H] 16a-hydroxydehydroepiandrosterone.

inhibition (8).

After

YoungLai

and

metabolites the

in

most

was

Solomon the

(12)

isolated

glucuronide

abundant

direct

metabolite

conversion

of

one

sulfate

to

The

presence

to

been

shows are

its

and

in

precursor

but

direct

liver

in

are (24)

for

able but

these

normal

human

sulfate

the

were

sterone

found

form in It

have steroid ment s similar of

in

remain

of

to

be

qualitatively

suggest

in that

YoungLai

and

formation

to

the

female

this

human

being

possibly

serves

identified

ketosteroid The

the

observe

and

and

only

This

Solomon, from

mainly that

the normal

of

present in

a

is

in

urine. who

injected

as

few

urine

contrast

were

is

disulfate females

This

unable

to

males

ketonic

patterns

parallels

conju-

and

quantitative

these

to

(7).

16-oxygenated

Preliminary

quantitatively male

of

16a-hydroxydehydroepiandro-

pattern

urine.

even

(8),

and/or

precursors

were

fraction.

is

same

study

compound

study

urine, of

which

interest

has

sulfates

16P-hydroxydehydroepiandrosterone

urine,

sulfates

steroid

steroids

parent

in

disulfate

the

system present

steroid

discounted.

identified in

16a-hydroxy-

The 19 main

the

Human

studied.

in which

the

(23).

epimerizing

C

(22).

dehydroepi-

epimerize

ketonic

be

quantities

in

estriol

of

for liver

16-hydroxylated

minor

infant

is

demonstrated

dehydroepiandroster-

steroids.

urine,

not

conjugates

samples

gated

other

must

steroids

monosulfate

the

the

fraction

sulfate

to

This

16-hydroxylation

steroids All

have

fetal

this

neutral

16-oxygenated

for

ketosteroid

sulfate

16P-hydroxylase

16a-hydroxydehydroepiandrosterone. as

et al.

radioactive

and

16P-isomer

several

present

--

demonstrated

demonstrated

that

injected

16a-

been

intestine

estrone not

both

has

placenta,

the

16-hydroxylated

16a-hydroxydehydroepiandrosterone of

androsterone

In

the

16a-hydroxydehydroepiandrosterone

Baulieu

of

of

fraction,

16a-hydroxyandrosterone.

the

most

measureare

the

C

very

results

demonstrate

16a-hydroxydehydroepiandrosterone

19

in

a

nonpregnant Analyses

during cate

249

STEROIDS

Sept. 1969 woman

of

the

pregnancy that

this

(12).

16”oxygenated

are

in

fraction

ketonic

progress

of

and

steroids

is

C

19 preliminary

very

steroids

in urine

results

prominent

indi-

in maternal

urine. ACKNOWLEDGMENTS We wish to thank Drs. W. Klyne and S. Solomon for generous gifts of reference steroids. We are grateful to Drs. H. Adlercreutz and T. Luukkainen for allowing us to use the gas chromatograph-mass spectrometer at the Department of Clinical University of Helsinki. Chemistry, The skillful technical assistance of Mrs. Marjatta Tevilin is gratefully acknowledged. This investigation was supported by grants from the National Research Council for Medical Sciences, and the Sigrid Jusklius Foundation. Finland,

1. 2.

Diczfalusy, 82 (1967). Easterling, land, M.V.

E.,

REFERENCES EXCERPTA MED.

INT.

CONGR.

W. E., Jr., Simmer, H.H., Dignam, and Naftolin, F., STEROIDS 8, 157

4.

zhindler, 28, 1189

5. 6.

zngiovanni, A.M., J. CLIN. INVEST. 41, 2086 (1962). Fotherby, K., Colas, A., Atherden, S.MT and Marrian,G.F., BIOCHEM. J. 66, 664 (1957). Shackleton, C.HyL., Kelly, R. W., Adhikary, P.M., Brooks, Harkness, R.A., Sykes, P.J. and Mitchell, F.L., J.W., STEROIDS 12, 705 (1968).

7.

8. 9.

YoungLai, Reynolds,

ET and Solomon, J.W., STEROIDS

10. 11.

Adams, J.B. and Shackleton, C.H.L.

12.

(1967). YoungLai,

13. 14. 15. 16. 17. 18. 19.

Siiteri,

Wong, and

K. J.,

Frank-

Magendantz, H.G. 24, 1155 (1964).

and

Ryan,

W.J., (1966).

132,

3.

A. E. (1968).

and

SERIES

P.K.,

S., 3,

M.SyF., Mitchell,

J.

CLfk.

ENDOCRINOL.

J.

CLIN.

ENDOCRINOL.

BIOCHEMISTRY 77 (1964). THE F.L.,

LANCET, STEROIDS

6, -

2040

C.

(1967).

1163 (1968). lo, 359 -

E.V. and Solomon, S., BIOCHEMISTRY 7, 1881 (1968). Janne, 0. and Vihko, R., SCAND. J. CLlN. LAB. INVEST. 23, Suppl. 108, 40 (1969). Janne, 0. and Vihko, R., ANN. MED. EXP. FENN. 46, 301 (1968). Laatikainen, T. and Vihko, R., EUROPEAN J. BIOCHEM. submitted for publication). (1969, Sjovall, J. and Vihko, R., STEROIDS 7, 447 (1966). ACTA ENDOCRINOL. Suppl: 109 (1966). Vihko, R., Laatikainen, T. and Vihko, R., STEROIDS (1969, in press). Gustafsson, J.-A., Lisboa, B.P. and Sjbvall, J., EUROPEAN J. BIOCHEM. 6, 317 (1968).

STEROIDS

250

20. 21. 22.

23. 24. 25.

14:3

T., Vainio, J. and Vihko, R., Jgnne, O., Laatikainen, 13, 121 (1969). STEROIDS ENDOCRINOL. 57, 247 Vihko, R., ACTA Sj&all, J. and (1969). Baulieu, E.-E., CorpLchot, C., Dray, F., Emiliozzi, R., Lebeau, M.-C., Mauvais-Jarvis, P. and Robel, P., RECENT PROGR. HORMONE RES. 21, 411 (1965). Slaunwhite, W.R., Karsay, ?%.A., Holmer, A., Sandberg, A.A. and Niswander, K., STEROIDS a, Suppl. II, 211 (1965). BIOPHYS. Dahm, K., Lindlau, M. and Breuer, H., BIOCHIM. ACTA 159, 377 (1968). 16a-hydroxyandrosterone, 3a, 16aSystematic nomenclature: -dihydroxy-5e-androstan-1 ‘I-one; 16a-hydroxyepiandrosterone, 3p, 16a-dihydroxy-5e-androstan-17-one; 16a-hydroxyetiochola3a, 16a-dihydroxy-S/3-androstan-17-one; 16a-hydroxydenolone, 3p, 1 ba-dihydroxyandrost-5 -en-l 7-one; hydroepiandrosterone, 16P-hydroxydehydroepiandrosterone, 3p, 16P-dihydroxyandrost-5-en-17-one; dehydroepiandrosterone, 3f3-hydroxyandrost-5-en-l 7-one; 3,16a-dihydroxyestra1 ba-hydroxyestrone, -1,3,5 (lo)-trien-17-one; estriol, estra-1,3,5 (lO)-triene-3, 16a, 17P-trial.