Sterol composition of the steryl sulfate fraction in human feces

417

STEROL COMPOSITION

OF T H E S T E R Y L

SULFATE FRACTION IN

HUMAN FECES. Bile Acids Peter Eneroth Department

and S t e r o i d s

196.

and Ernst Nystr8m.

of C h e m i s t r y ,

Kar01inska

Institutet

S t o c k h o l m 60, S w e d e n .

Received January 29, 1968

ABSTRACT Cholesterol, cholestanol, lathosterol, campesterol, campestanol, stigmasterol, ~-sitosterol and ~-sitostanol, h a v e b e e n i d e n t i f i e d i n the s t e r y l s u l f a t e f r a c t i o n f r o m feces of h e a l t h y s u b j e c t s o n a free diet. E v i d e n c e was also o b t a i n e d for the p r e s e n c e of b r a s s i c a s t e r o l a n d fuc o s t e r o l . No 3 ~ - h y d r o x y - 5 ~ - s t e r o l s c o u l d be found. The a m o u n t of c h o l e s t e r y l s u l f a t e was e s t i m a t e d to at l e a s t 8 5 % of the e n t i r e s t e r y l s u l f a t e f r a c t i o n . INTRODUCTION In the c o u r s e

of a q u a n t i t a t i v e

c r e t i o n of c h o l e s t e r y l steryl

sulfate

f r a c t i o n was

pounds with thin-layer identical

sulfate

in human feces

to that of c h o l e s t e r y l the r e s u l t s

spectrometric

(GC-MS)

solvolysis

(TLC)

sulfate.

of a gas

the

of the s t e r y l

mobilities

The present

chromatographic-mass

s t u d y of the c o m p o u n d s

EXPERIMENTAL

(1)p

f o u n d to c o n t a i n m a n y com-

chromatographic

paper reports

upon

s t u d y o f the ex-

sulfate

liberated

fraction.

PROCEDURE

A d e t a i l e d d e s c r i p t i o n of the m e t h o d s u s e d has b e e n g i v e n i n a r e c e n t r e p o r t (I). B r i e f l y , the p r o c e d u r e in-

418

ST ER O ID S

11:4

c l u d e s the f o l l o w i n g steps: A c h l o r o f o r m / m e t h a n o l I:~4C_. (v/v) e x t r a c t o f h u m a n feces to w h i c h c h o l e s t e r y l - ~ s u l f a t e h a d b e e n a d d e d was e v a p o r a t e d a l m o s t to d r y n e s s . The r e s i d u e was p a r t i t i o n e d b e t w e e n l i g h t p e t r o l e u m a n d 0.2 M a m m o n i u m c a r b o n a t e in 65% a q u e o u s e t h a n o l . The a q u e o u s p h a s e was c o n c e n t r a t e d and the r e s i d u a l s o l u t i o n was made 2 M w i t h r e s p e c t to p y r i d i n e s u l f a t e a n d was ext r a c t e d w i t h c h l o r o f o r m . C h l o r o f o r m was e v a p o r a t e d a n d the r e s i d u e was c h r o m a t o g r a p h e d o n a c o l u m n of m e t h y l a t e d S e p h a d e x G - 2 5 . E l u t i o n o f this c o l u m n w i t h c h l o r o f o r m / m e $ ~ a nol 1:4 y i e l d e d a f r a c t i o n c o n t a i n i n g the c h o l e s t e r y l - 4 - ' ? C c h o l e s t e r y l s u l f a t e . T h i s f r a c t i o n was f i l t e r e d t h r o u g h an A m b e r l y s t - 1 5 i o n e x c h a n g e r in the a m m o n i u m f o r m i n c h l o r o f o r m / m e t h a n o l 1:4. The e f f l u e n t was e v a p o r a t e d and the r e s i due was c h r o m a t o g r a p h e d o n a c a r b o x y m e t h y l S e p h a d e x L H - 2 0 c o l u m n in the a m m o n i u m f o r m i n c h l o r o f o r m / m e t h a n o l ~:I. L a b e l e d f r a c t i o n s w e r e pooled, the s o l v e n t was e v a p o r a t e d and the r e s i d u e was s o l v o l y z e d in r e f l u x i n g d i o x a n e for Shree hours, and t h e n n e u t r a l i z e d w i t h the A m b e r l y s t ion e x c h a n g e r . A f t e r e v a p o r a t i o n of the solvent, the r e s i d u e was c h r o m a t o g r a p h e d on a c o l u m n of S e p h a d e x L H - 2 0 in m e t h y l e n e c h l o r i d e / b e n z e n e 2:1 (2). The f r a c t i o n s w e r e a n a l y z e d by TLC in b e n z e n e / e t h y l a c e t a t e 7:1 a n d b y g a s - l i q u i d c h r o m a t o g r a p h y (GLC) on QF-I c o l u m n s (3,4). T r i m e t h y l s i l y l (TMSi) e t h e r s of s t e r o l s w e r e a n a l y z e d o n a c o l u m n of I% S E - 3 0 o n a c i d washed, d i m e t h y l d i c h l o r o s i l a n e t r e a t e d G a s C h r o m P ( A p p l i e d S c i e n c e L a b o r a t o r i e s Inc., S t a t e C o l l e g e , Pa., USA) w i t h an L K B M o d e l 9 0 0 0 Gas C h r o m a t o g r a p h - M a s s S p e c t r o m e t e r ( L K B - p r o d u k t e r , S t o c k h o l m , S w e d e n ) . R e t e n t i o n times are g i v e n r e l a t i v e to that of c h o l e s t e r y l T M S i e t h e r ( R R T v a l u e s ) . C o l u m n c o n d i t i o n s were: F l a s h h e a t e r t e m p e r a t u r e o o 290 , c o l u m n t e m p e r a t u r e 234 a n d h e l i u m f l o w 35 m l / m i n . T h e t e m p e r a t u r e of the m o l e c u l e s e p a r a t o r a n d the i o n s o u r c e w e r e 240 ° a n d 290 ° , r e s p e c t i v e l y . T h e e n e r g y of the b o m b a r d i n g e l e c t r o n e s was 2 2 . 5 eV a n d the i o n i z i n g c u r r 2 ~ t was 60 ~A. The v a c u u m in the a n a l y z e r tube was I-2 x 10 V m m Hg. M a s s s p e c t r a w e r e r e c o r d e d w i t h a s c a n s p e e d of 4-6 s e c o n d s (m/e 12-500). RESULTS After purification column,

the p o o l e d s t e r y l

spot o n TLC analysis; repetitive relative

on a c a r b o x y m e t h y l

development

mobilities

t i v e l y (I). A f t e r on S e p h a d e x LH-20,

sulfate

however,

Sephadex LH-20

f r a c t i o n gave r i s e

after dioxane

TLC r e v e a l e d

of c h o l e s t e r o l

solvolysis ,

two c o m p o u n d s w i t h and cholestanol,

c h r o m a t o g r a p h y o f the s o l v o l y z e d fractions

to one

containing

al-

the

respecsterols

April 1968

most

STEROIDS

exclusively

cholesterol

cholesterol

fractions,

were

however,

as s h o w n in fig.

1. This

subject

(out of the four

studied)

amount

steryl

sulfate

obtained

~0

of sterols

other

fraction.

during

eo

Fig.

the a n a l y s i s

so

obtained

contained

sterols

lative

419

(lj2). E a r l y mixtures

sample was having

f r o m the

the l a r g e s t

than c h o l e s t e r o l 2 shows

the mass

s h o w n in fig.

40

3'0

of

re-

in the spectra

I.

20

10

MINUTES

Fig. I. Gas c h r o m a t o g r a p h i c analysis on a 1% B E - 3 0 c o l u m n of T M S i ethers of sterols o b t a i n e d a f t e r solvolysis of a p u r i f i e d h u m a n fecal steryl s u l f a t e f r a c t i o n and s u b s e q u e n t r e m o v a l of most of the c h o l e s t e r o l . M a s s s p e c t r a w e r e r e c o r d e d at times n o t e d in the c h r o m a t o g r a m by v e r t i c a l lines. T h e s e p o s i t i o n s are s u b s e q u e n t ly r e f e r r e d to as G L C - p e a k s . The mass s p e c t r a are s h o w n in Fig. 2. For i d e n t i f i c a t i o n s see text. The mass 1.00)

spectrum recorded

h a d peaks 247,

at m/e 458,

275,

255,

TMSi

ether of c h o l e s t e r o l

460,

445,

403,

213,

355,

and

306,

443,

129 due

for G L C - p e a k 368,

353,

2 (RRT =

329,

301,

to f r a g m e n t a t i o n

(3, 5 and 6). The p e a k s 305,

230,

216,

of the at m/e

and 215 and their

420

ST ER O ID S

relative mass

abundance

spectrum

3). These sents

were

the same as those

of a u t h e n t i c

data s t r o n g l y

a mixture

cholestanol.

(cf. ref.

cholestanyl

suggest

of the T M $ i

is c o m p o s e d

at m/e 458,

213 are those ether

ether

ryl TMSi

pattern.

been ruled

368,

353,

Although

and TMSi

out by TLC

(3-5).

spectrum

traces

ether

explain

found at m/e 470

p e a k at m/e

TMSi

3 (as

fragTMSi

j u d g e d by the

of yet

of G L C - p e a k

of the

another

ste-

/kS-struc 3 is that

to

of 2 4 - m e t h y l - c h o l e s t a - 5 , The p r e s e n c e

support

(molecular

ether

intensity

the c o m p a r a t i v e l y

which

frag-

229 and

of c h o l e s t e r y l

the p r e s e n c e

time

The

3

of l a t h o s t e r y l

of a p r o m i n e n t

(brassicasterol).

129. F r a g m e n t s

247,

the 3 - t r i m e t h y l s i l y l o x y -

for the TMSi

compound would

255,

the h i g h r e l a t i v e

The r e t e n t i o n

22-dien-3~-ol

2) G L C - p e a k

compounds.

345,

in G L C - p e a k

129 suggests

ether w i t h

(fig.

of the l a t h o s t e r y l

at m/e 329),

be e x p e c t e d

m/e

(fig.

2 repre-

epicholestanyl

of s e v e r a l

found in the mass

a feature

p e a k at m/e

ture

443,

are still p r e s e n t

fragment

ether

of c h o l e s t e r o l

spectrum

(7). The p r e s e n c e

129 is not mentation

TMSi

1).

(RRT = 1.12)

TM$i

that

had p r e v i o u s l y

As s e e n from the mass

ments

f o u n d in the

that G L C - p e a k

ethers

The p o s s i b i l i t y

ether was p r e s e n t

11:4

ion),

this 455

of this

large

p e a k at

interpretation (M-15),

386

are

(M-84),

38o (M go), 372 (M-(97+I)), 365 (M(9o+15)), 343 (M-(125+ 2)), 341 (M-129), 337 (M-(90+43)) and 253 (M-(90+125+2)). This

fragmentation

pattern

is in a g r e e m e n t

with

the

April 1968

ST ER O ID S

suggested C22-C23 peaks

fragmentation

double

bond

are found

assume

that

m/e 83 and ether

of a steryl

69 and

fragments

139 in the mass

(4). The

rated C28

presence

steryl

and a methyl

TMSi

group

the o c c u r r e n c e

TMSi

ether w i t h

(4). In the lower mass

at ~ e

these

421

125.

spectrum

in the side

of peaks

to

to the peaks

at

of s t i g m a s t e r y l

in G L C - p e a k

ether w i t h

end p r o m i n e n t

It is t e m p t i n g

correspond

a

3 of a C5-C6

a C22-C23

chain

at m/e 472,

TMSi

satu-

double

bond

is s u s p e c t e d

from

457

(M-15),

388

(M-

84), 374 (M-(97+1)) and 257 (M-(125+90)). The mass reveals 289,

tion, 276,

fragments

261,

tation

spectrum

255,

pattern peaks

257,

campestanyl Mass

at m/e

213,

472,

and 129 w h i c h

recorded

and 215 w h i c h

TMSi

367,

represent

TMSi

ether

In addi-

417,

306,

the p r e s e n c e

5 (cf.

spectrometry

of G L C - p e a k

6 (RRT = 1.39)

containing

fragments

351,

345,

211,

139,

129,

and 83, w h i c h

spectrum

other

terpreted.

peaks The

since

unsaturated

343,

are also

are

fragment

3~-TMSi

ether

253,

those

ether

seen but at m/e

fig.

at m/e 484,

255,

of the TMSi

fragments

this

271,

315,

the fragmen-

(3,5).

459,

indicate

343,

in G L C - p e a k

372,

terest

382,

at m/e 474,

379,

Many

457,

5 (RRT = 1.29)

ether

a spectrum

in a mass

for G L C - p e a k

of c a m p e s t e r y l

were

216,

shown

243,

305,

of

3).

469, 215,

yielded 394, 213,

to be a n t i c i p a t e d

of s t i g m a s t e r o l

cannot

be easily

386 is, however,

is k n o w n

as typical

derivatives

of C28

(4). in-

of infor ring-

and C29

422

S T E R O ID S

sterols w i t h a C 2 4 - C 2 8 47 ° and 484,

respectively)

The major peaks (RRT = 1.61)

213,

at m/e 486,

of G L C - R e a k

383,

306,

(3,5). 471,

spectrum

Thus

396,

typical

381,

of G L C - p e a k

357,

8 (RRT = 1.67)

290,

7

of the T M S i

fragments 329,

275,

were 255,

can be f o u n d in the mass

such as those

305,

weights

(5).

same peaks

are more p r o m i n e n t 398,

(molecular

are those f o u n d in the s p e c t r u m

and 129. The

spectrum

bond

in the mass

ether of ~ - s i t o s t e r o l recorded

double

11:4

257,

but

other

at m/e 488,

230,

217,

216,

fragments 473,

431,

and 215 re-

presenting the fragmentation p a t t e r n of the T M S i ether derivative peaks

typical

rivative 394,

of ~ - s i t o s t a n o l

379,

371,

The d i f f e r e n t trace

amounts

are l i b e r a t e d steryl

mass

prostanyl position sence

spectra

and

spectra

ether.

A mass

In addition, ether de-

at m/e 484,

229 and 228

in fig.

2 also

469,

(5). s h o w that

than those m e n t i o n e d

of p u r i f i e d h u m a n

fecal

no i n t e r p r e t a t i o n

could

of G L C - p e a k s

10 (RRT = 2.64).

of G L C - p e a k

of this

i.e.

compounds

Furthermore,

3).

of the T M S i

281,

2 was i n v e s t i g a t e d

TMSi

4 (RRT = 1.17),

The r e g i o n

for the p r e s e n c e

spectrum recorded

I did not give

evidence

in front of coat the

for the pre-

derivative.

To o b t a i n a r o u g h importance

296,

upon solvolysis

be made of the mass

of G L C - p e a k

are seen:

343,

of o t h e r

sulfates.

9 (RRT = 2.02)

fig.

of the f r a g m e n t a t i o n

of f u c o s t e r o l

386,

(of.

estimation

of the d i f f e r e n t

of the q u a n t i t a t i v e

sterols

in the s o l v o l y z e d

April 1968

STEROIDS

423

I00 P-LC PEAK 2

/

% 50

0

50

100

150

.,:..~..,11.. j:,-, J,. L~ . J . . . . . d . 200 250 300 role

:

:

,L

,

•

.

i

. . . .

m

,

,

350

400

450

500

350

400

450

5OO

" '450 . . . .

5oc

500

100

I

h

GLC PEAK 3 % so

0

•

50

100

150

200

250 m le

300

100 GLC PEAK 5 % 50

I -, , . . 3 . , ~ L ....

5"0 . . . .

100" "

"150 . . . .

260 . . . .

2SO . . . . m/e

3t~0 . . . .

350 . . . .

, "400" "

100

150

200

250

300

350

400

450

,L IJM u~L 150

200

250 m/e

300

350

400

450

100 G L C PEAK 6

% so

C

5O

role

100 GLC PEAK 7 %50

,,

0 "

"

/ .

sO

Ibo

|

L

500

100

t

G L C PEAK 8 % 50

50

100

150

200

250 m/e

300

350

400

450

Fig. 2. Mass spectra o b t a i n e d during the GC-MS analysis shown in fig. 1.

500

424

ST ER O ID S

11:4

lOO CHOLESTANYL TMSi ETHER % SO

I

, _IL. J, J~ .~,..,.[~,....L[I.~ . . . .

S'O

100

"

F i g . 3. M a s s ether. steryl a GLC

"

sulfate

the

Sephadex

and

cholestanol

sterols. higher

The

than

of a u t h e n t i c

from

was

before

made

column

(which

for

relative 5% as of

the

of

the

400

90%

(cf.

on

subjects

the

on

Cholesterol on of

ref,

the G L C

the

cholestanol

by TLC

TMSi

four

separated

of

450

fractionation

at l e a s t

sterols

the

performed.

not

amount

judged

350

cholestanyl

each

was

were

accounted

fractionation

300

fraction

LH-20

used)

ILLL

250 m/e

=00

spectrum

analysis

phases

"1,¢0 . . . .

total

was I)

Sephadex

not

after LH-20

columns.

DISCUSSION The

presence

was

first

the

isolated

scopy

and

liberated in

the

mass

of a s t e r y l

reported

cholesteryl

showed upon

present

solvolysis. study

it l i k e l y

are

data.

on S e p h a d e x that

the

et

al.

sulfate

(by TLC

spectrometric

matography

by Moser

sulfate

a n d NM_R) The based The

LH-20

compounds

in human

(8)

by

who

identified

infrared

that

feces

spectro-

cholesterol

identifications on

was

made

chromatographic

and

results

obtained

by

chro-

(2)

and

with

TLC

(1)

studied

with

GC-MS

make

belong

April 1968

STEROIDS

to a g r o u p ~9

of m o n o h y d r o x y

series.

tures

of c o m p o u n d s

tively

identified

parison the

Although

sterols

of the C27 , C28

spectra

were

a n d in s o m e fucosterol

c o u l d be m a d e

interpretations

Mass

mass

spectrometry

with

also

would

and TLC would

amounts

probably

was

not

if for

TMSi

the

steryl

sulfate

fraction

was

at l e a s t

tal

sterols.

the

sterols

(not

composition

precise

presence

sterols

no

shows

steryl

of was

i n this

fecal

steryl

the r e l a t i v e made.

such that

compound the m a j o r

monosulfate

a 3~-hydroxy-~

fraction

was

has

of the

and

Since

study)

may

sulfate

the

influence

of

no

the

to s h o w

steryl

Thus

dieta-

fraction,

attempts

the

sulfate

the p r e s e n t

in the h u m a n

either

cam-

~-sitostanol

15%.

found.

in

to-

campesterol,

in the

sterols

85%

proportions

Despite

of 3 ~ - h y d r o x y - 5 ~ - s t e r o l s

fraction, study

~-sitosterol

of the r e m a i n i n g

of the

evaluation

different

lathosterol,

determined

present.

of c h o l e s t e r o l

amount

ry

but

and in e a c h of the

the

for m o s t

between

sterol

analyzed

accounted

times.

significant

had been

samples

stigmasterol,

com-

compounds,

of a s t e r o l

fecal

pestanol,

tenta-

no

retention

instance

ether

the p r e d o m i n a n t

Cholestanol,

the

distinguish

derivative

shown

(i.e.

of a u t h e n t i c

with GLC

and

for mix-

and brassicasterol)

agree

of e p i c h o l e s t e r y l

Cholesterol four

have

recorded

instances

spectra

a 3 a - ~ 5- a n d a 3 ~ - A 5 T M S i GLC

425

fecal

a 3~-hydroxy-Sa-,

5- or a 3 ~ - h y d r o x y - 5 ~ - ~ 7 - s t e r o i d

nucleus

structure. Since

coprostanol

and

the

corresponding

plant

sterol

426

ST ER O ID S

derivatives sterols stanol

are

in m a n may

been

origin

found

connection

it

have

the

shown

lathosterol, germfree have

liver

that

the

plant

have

been

structural it

as w e l l

as

tion

substrates

as

products plant

called

c a n be

sterols

in

sulfate formed

the

to be p r e s e n t

cholesterol

the

the

previously

have

been

This of

pig

In

liver. and

compounds

may

that

a

shown

cholesterol

latter

bile.

(12)

bile.

in guinea

and

(11)

from

action

companions

reaction

al.

recently

between

that

into

the

extra-

this

feces

human

one

of

sterols,

and Lieberman

through

copro-

In et

in

fecal

activity

of p l a n t

from

to

likely

excreted

must

Drayer

of

are

(10).

and methostenol

this

lack

sulfates

mucosa

resemblance

seems

this

that Gustafsson

similar

(13)

formed

sulfurylating

sulfates

Furthermore,

and Roy

so

some

of

cholesteryl

sterols

steryl

of i n t e r e s t

desmosterol

could

the

the

(7,9)

intestinal

sulfotransferase

to

animals

presence

rats.

by B a n e r j e e Due

and

microbiologically

although

in is

isolated

compound

(3)

indicate

intestinal has

the m a j o r

11:4

the

that

absorbed

also

func-

reaction case from

the the

intestine. The

metabolic

completely steryl hormone ryl be

known

sulfate

but

c a n be

sulfates

sulfates

significance been

directly

(14,15).

undergo

established.

it h a s

of

sulfates

demonstrated

transformed

Whether

similar

steryl

or n o t

that

into the

transformations

is

in-

chole-

steroid

other

ste-

remains

to

April 1968

ST ER O I D S

427

ACKNOWLEDGEMENTS The skilful technical assistance of Miss Kerstin Persson is gratefully acknowledged. This work has been supported by grants from Stiftelsen Svensk NXringsforskning and from SXllskapet fSr Medicinsk Forskning. REFERENCES I.

3.

Eneroth, press. Eneroth, 144, 149 Eneroth,

4.

Eneroth,

2.

P., and Nystr8m,

E., STEROIDS

(1968),

in

P., and Nystr8m, E., BIOCHIM. BIOPHYS. ACTA (1967). P., Hellstr8m, K., and Ryhage, R., J. LIPID

RES. ~, 245 ( 1 9 6 4 ) . P., Hellstr~m,

K., and Ryhage, R., STEROIDS

~, 707 ( 1 9 6 5 ) . 5. 6.

Knights, Diekman,

B.A., J. GAS CHROMATOG. ~, 273 (1967). J., and Djerassi, C., J. ORG. CHEM. 32, 1005

7.

Gustafsson, B.E., Gustafsson, J.-A., and Sj~vall, J., ACTA CHEM. SCAND. 20, 1827 (1966). Moser, H.W., Moser, A.B., and 0rr, J.C., BIOCHIM. BIOPHYS. ACTA 116, 146 (1966). Danielsson, H., ADVANCES IN LIPID RESEARCH Eds. Kritchevsky, D., and Paoletti, R., Academic Press, Inc., New York, N.Y. 1963. BostrGm, H., and Wengle, B., ACTA ENDOCRINOL. 56,

(1967). 8. 9. 10.

691 ( 1 9 6 7 ) . 11. 12. 13. 14. 15. 16.

o

Gustafsson, B.E., Gustafsson, J.-A., and Sj8vall, J., to be published. Drayer, N.M., and Lieberman, S., BIOCHEM. BIOPHYS. RES. COMMUN. 18, 126 (1965). Banerjee, R.K., and Roy, A.B., BIOCHIM. BIOPHYS. ACTA 1~7, 213 (1967). Roberts, K.D., Bandi, L., Calvin, H.Ie, Drucker, W.D., and Lieberman, S., BIOCHEMISTRY ~, 1983 (1964). Gurpid~, E., Roberts, K.D., Welch, M.T., Bandy, L., and Lieberman, S., BIOCHEMISTRY ~, 3352 (1966). The following systematic names are given to sterols referred to in this report by trivial names: cholesterol, cholest-5-en-3~-ol; epicholesterol, cholest-5-en3~-ol; cholestanol, 5~-cholestan-3~-ol; epicholestanol, 5a-cholestan-3~-ol; coprostanol, 5~-cholestan-3~-ol; lathosterol, 5a-cholest-7-en-3~-ol~ brassicasterol, 24~-methyl-cholesta-5,22-dien-3~-ol; campesterol, 24~methyl-cholest-5-en-3~-ol; campestanol, 24a-methyl5a-cholest-3~-ol; stigmasterol, 24a-ethyl-cholesta-5, 22-dien-3~-ol~ ~-sitosterol, 24~-ethyl-cholest-5-en3~-ol; ~-sitostanol, 24a-ethyl-5~-cholestan-3~-ol; fucostorol, 24-ethylidene-cholest-5-en-3~-ol.