Quantification of cholesteryl sulfate and neutral sterol derivatives in human feces after purification on lipophilic sephadex gels

187

QUANTIFICATION

OF

DERIVATIVES

CHOLESTERYL

IN HUMAN

FECES

PHILIC Bile Peter Department

SULFATE AFTER

SEPHADEX

Steroids

Eneroth

and

Ernst

Stockholm

Received April 25,

ON

LIPO-

188

Nystrom

Karolinska

60,

STEROL

GELS

and

Chemistry,

NEUTRAL

PURIFICATION

Acids

of

AND

Institutet,

Sweden.

1967

ABSTRACT The sterol sulfate fraction in human feces has been analyzed by an isotope dilution technique. In the four 14 to 85 mg of cholesteryl sulfate were subjects studied, found in a one day portion of feces. This corresponded to 2.2-3.9s of the total cholesterol output. In contrast, pronounced variations between the subjects were found upon analysis of the neutral fecal cholesterol metabolites. INTRODUCTION The has

presence

been

shown

by Moser

et

With

(1).

steryl dered

regard

and

tes

in

feces

viously

used

have

not

but to

in

of value

tively

cholesteryl

in patients

al.,

sulfate it

of

no the

been

from for

with

possible

to measure it

normal the

designed

this

persons.

determination to

data

include

were

cholesterol

of the

of

studies

compound

Since

feces

diseases

importance

balance

to other

in human

neurological

quantitative

cholesterol

to relate

sulfate

reported cholewe

consi-

quantitametaboli-

techniques fecal labile

pre-

sterols choleste-

188

11:2

STEROIDS

ryl

sulfate

order

to

isolate

quantitation. procedures gels

a novel

The

the

procedure

cholesteryl

method

utilizing

has

the

been

developed

sulfate

prior

is based inert

on

in to

its

chromatographic

lipophilic

Sephadex

(2). MATERIALS

AND

METHODS

chloroform (stabilized with 1% ethanol, ADA, Solvents: Stockholm, Sweden) and methanol (p.a_, Merck AG., Darmwere used as supplied. Light petroleum stadt, Germany) 1,2-dichloromethane (b.p. 40-6Oo), benzene, dioxane, and pyridine were purified according to standard proon PorapakR Q cedures 3 D When gas chromatographed 4 (Waters Assoc., Inc., Framingham, Mass., columns I! USA) the purified solvents appeared to be at least 99% was tested for peroxides immediately bepure. Dioxane fore use* Reference substances. Sterols and steroid derivatives were supplied by Ikapharm, Ramat-Gan, Israel and by Southeastern Biochemicals, Morristown, Tenno, USA. The purity of the compounds was checked with gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) Cholesterol-4-14C (specific activias described below. was supplied by the Radiochemical Centre, ty 20 mC/mM) Cholesteryl sulfate, with and withAmersham, England. out 14C-labeling, was synthesized by the procedure described by Kornel et al. (5) and purified by the procedure described below. The isolated ammonium salt was crystallized from methanol by the addition of chloroform. It melted with decomposition at 189-193O; reported 197201° (6) and 185~ 7). The infrared spectrum of this compound (fig. 3) i cf. ref. (6)), was determined on a Solvolysis in dioxane Perkin-Elmer 221 Spektrophotometer. (8) yielded a fraction which, according to TLC and GLC, was pure cholesterol. Labeled cholesteryl sulfate was analyzed by TLC. Autoradiography and subsequent spraying with sulfuric acid revealed one spot with a mobility corresponding to that of cholesteryl sulfate. Pyridine sulfate for the extraction of steroid sulfates was prepared by titrating aqueous H2SO4 with an equivalent amount of pyridine to form a 4M solution of pyriThe stock solution was titrated so that a dine sulfate. 0.2 M solution had a pH of 4.0, Ammonium carbonate for the extraction of acidic material (Merck AG., Darmstadt, Gerwas p.a. grade 7-see below many).

Feb. 1968

STEROIDS

189

Trimethylsilyl (TMSi) ethers for GLC were prepared by adding 0.3 ml pyridine, 0.1 ml hexamethyldisilazane and 0.05 ml trimethylchlorosilane (Applied Science Laboratories Inc., State College, Pa., USA) to 5 mg of sterol material. After 30 min the reaction mixture was taken to dryness under a stream of nitrogen and the residue dissolved in dry light petroleum and analyzed within an hour. Saponification of fatty acid esters of sterols was accomplished by leaving 10 mg of esters in a solution of 1.3 ml dry dioxane and 0.35 ml IM potassium hydroxide in methanol for 16-20 hours at room temperature. The reaction mixture was then extracted with light petroleum (Lp). The extract was subsequently washed with several portions of 60% aqueous ethanol, each portion l/20 of the volume of the Lp. Each washing portion was reextracted with ten times its volume of fresh Lp. The Lp phases were combined and taken The residue was stored at 3O until analyzed. to dryness. Solvolysis of the steroid sulfates was accomplished by refluxing in dioxane (1 mg/ml) for three hours. The cooled solution was filtered through a column of 10 g Amberlyst-15 in the ammonium form packed in CHC13/CH30H I:4 (volume/volume as will be used throughout this paper). The ion exchanger was then washed with 25 ml of the chloroform/ methanol mixture. The eluate was taken to dryness and the residue stored at 30 until further analyzed, Radioactivity determination. A gas-flow counter was used for the monitoring of chromatographic effluents. For quantitative measurements, a Packard Tri-Carb liquid scintillation spectrometer (model 4322), was used with the liquid scintillator described by Bray (9). The samples were evaporated under a stream of nitrogen in the counting vials and were then dissolved in the liquid scintillator. Each determination was made in duplicate, counting at least 10000 impulses per sample. To correct for quenching, two more sa ples were determined after the addition $ C sulfate. of cholesteryl-4-l Lipophilic gel materials for chromatography. Methylated Sephadex G-25 (bead form, Pharmacia, Uppsala, Sweden) was prepared as described previously (10). This material is designated G-25-36. Similarly, G-25-42 denotes a Sephadex G-25 gel having a methoxyl group content of approximately 42%. The preparation of this derivative will be described separately (11). To prevent floating of the G-25-42 in chloroform, the gel bed was kept down with a porous teflon membrane, and was eluted under nitrogen pressure (ca. 100 mm Hg). Sephadex LH-20 (kindly given to us by Dr. B. Gelotte, Pharmacia, Uppsala, Sweden) was used as supplied and as a starting material for the synthesis of a carboxymethyl derivative (designated CM-Sephadex-LH-20 (11). The gel used in this study had an ion exchange capacity of 0.24 meq/g and approximately the same swelling properties as unsubstituted Sephadex-LH-20.

190

STEROIDS

The packing of a column of a coloured substance.

was

11:2

checked

by

chromatography

Ion exchange chromatogra h D Amberlyst-15 (batch no0 625), with a capacity of 4.9 * me given to us by g, was kindly Rohm and Haas Co., Philadelphia 5, Pa., USA. A column of 2.5 g Amberlyst-!j-H+ in chloroform/methanol I:4 was converted into the ammonium form by elution with 10 ml of 25% ammonium hydroxide/95$ ethanol I:1 and subsequent washing with chloroform/methanol 4:l until the eluate was neutral. CM-Sephadex-LH-20 was converted into its ammonium form by washing the gel on a sintered glass funnel with the ammonium hydroxide solution described above followed by methanol and chloroform/methanol 4:l. Thin-layer chromatography. TLC analyses of the steryl sulfates were made with the solvent system described by Wusteman et al. (12): benzene/ethyl methyl ketone/ethanol/waFor lipid class separations the two-stage deter 3:3:3:1. veloping technique described by Freeman and West (13) was were run in benzene/ethyl acetate mixtures used. Sterols as originally described by Van Dam (14). Compounds were located by spraying with 80s sulfuric acid saturated with K2Cr203 followed by heating. Gas-liquid chromatography. The stationary phases QF-1 (3%) and SE-30 (1%) were used on acid-washed Gas-chrom P (Applied Science) silanized with dimethyldichlorosilae as decolumn conditions scribed previously (15). Approximate temperature 290° were: flash heater column temperature 2.0 kp/cm '2 D An argon ioniza234O, argon inlet pressure tion detector operated at 240° was used, The number of theoretical plates calculated for cholesterol were: &F-l: 2000. Retention times are given relative to 1800; SE-30: that of cholesterol (RRT-values). EXPERIMENTAL

PROCEDURE

Preparation of crude fecal extracts ( see fig. I), Feces from two healthy females and two healthy males (age: 2028) were collected during one day. The samples were hoAbout 1 +C of mogenized in 500 ml of distilled water. cholesteryl-4-14C sulfate was added as internal standard during the homogenization. The homogenate was lyophilized and the residue suspended in chloroform/methanol 1:l. The into a large chromatographic suspension was transferred tube and the solids were allowed to settle. The resulting packed material was eluted as an ordinary chromatographic column with chloroform/methanol 1: 1 until no more colored material could be eluted. This usually required 5-6 1. The weight and activity were determined on small aliquots, Usually 1 1 of each extract was taken for subsequent work-up.

Feb. 1968

191

STEROIDS

HOMOGENIZED. THE AND

RESIDUE

LYOPHILIZED OF

THE

0.2 M AMMONIUM

FECES

EXTRACT

IS EXTRACTED

IS PARTXTIONED

CARBONATE

IN 65

WITH BETWEEN

CHC13/CH30H LIGHT

I:,

(v/v).

PETROLEUM

% ETHANOL

r

1

PARTITION:

I STEROID

IN CHC13

KETONES

2 M PYRIDINE I

STERYL

(SK)

SEPHADEX

ESTERS

IN

(SE)

I GLC jGLC1

(9

1

G-25-36

CHC13/CH30H

1:4

I

I STEROLS

PYRIDINE SULFATE PHASE DISCARDED

SULFATE/

AMBERLYST-

sArFICAT1oN GLC

IN

15-NH4+

CHC13/CH30H

1:4

I CM-SEPHADEX-LH-20-NH4+ IN CHC13/CHJOH

Figure 1. Procedures lipid extracts.

employed

in

the

analysis

4:1

of

fecal

The solution was concentrated under reduced pressure at room temperature to about 25 ml. Fifty ml of 0.2M ammonium carbonate in aqueous 65% ethanol were added and after standing at room temperature for 15 min the extract was transferred to a separatory funnel., Successive rinsing with small portions of the ammonium carbonate solution and light petroleum (Lp) gave a final mixture in the funnel of 100 ml of the ammonium carbonate solution and 300 phase was then extracted successiml of Lp. The alkaline vely with 300-ml portions of Lp until no colored material The combined Lp phases were then appeared in the Lp phase. washed twice with 30-ml portions of the ammonium carbonate solution and then with 30-ml portions of 65% aqueous ethanAt this stage three additional washings ol until neutral. phase was reextracted with 300 ml were made. Each washing Lp phases aliquots were withof Lp. From the combined drawn for weight and radioactivity determination. The remainder was evaporated and the residue (Fraction N) kept analyzed. at 3O until

192

STEROIDS

The combined aqueous phases, 300-400 ml, were concentrated under a stream of nitrogen at room temperature and atmospheric pressure to one third or less of the original volume. An equal volume of 4M pyridine sulfate was added to the residue and the resulting solution was transferred to a separatory funnel, carefully rinsing with 2M pyridine sulfate and chloroform. The aqueous phase was extracted once with twice its volume and then twice with equal volumes of chloroform. Occasionally a dark precipitate was formed during the extraction. This material and the aqueous phase were discarded. The chloroform extracts were combined and the weight was determined on an aliquot. Evaporation of the solvent at room temperature under reduced pressure yielded a residue which contained traces of pyridine. This material was stored at -IO0 until further analyzed (see fig. 1). The chloroform/methanol I:1 extract from each subject was worked up at least in duEach light petroleum fraction was analyzed furplicate. ther at least twice. Chloroform extracts were analyzed in triplicate or more. Chromatographic purification of the light petroleum extract (Fraction N). This was accomplished as described Each fraction was taken to dryness under infig. 16 ) nitrogen and w:s weighed. Steryl esters were located by TLC using 100 @g from each fraction. Fractions containing steroid ketones were located by a combined TLC and GLC analysis. The pooled steryl esters were saponified (see above) and the sterols thus liberated were quantitatively determined by GLC of the steryl-TMSi derivatives. Steroid ketones were combined and analyzed as such. purification of the chloroform extract Between 500 and 1000 mg of the material in the chloroform extract, dissolved in 1.0-1.5 ml of chloroform/methanol I:4 was applied to a column-of 130 g Sephadex G-25-36 packed in the same solvent. When the void volume had been eluted, IO-ml fractions were collected until a total effluent of one total bed volume (=I.00 TBV) had been eluted from the column. The fractions were assayed for radioactivity and the labeled material eluted between about 0.65 and 0.73 TBV was combined and slowly filtered through a column of 2.5 g of Amberlyst-15-NH4+ packed in chloroform/methanol I:4 (column dimensions: portion of 50 ml chloroform/ 0.55 x I8 cm). An additional methanol I:4 was used to rinse the Amberlyst column. The combined filtrates were taken to dryness at room temperature under reduced pressure and the residue was applied in as small a volume as possible (usually 0.3-0.5 ml) to a column of 19 g CM-Sephadex-LH-20-NH4+ in chloroform/methanol 4:1 (column dimensions: 1.46 x 46,5 cm). When the void volume had been eluted, IO-ml fractions were collected. Labeled material, eluted between 1.1 and and evaporated at room temperature 1.5 TBV, was pooled

11:2

STEROIDS

Feb. 1968

193

under reduced pressure. The residue was analyzed by TLC and for absence of free sterols by GLC. Solvolysis was performed with l-3 mg of material from the radioactive fraction whereu on the solution was P column. filtered through an Amberlyst-15-NH4 The resulting material was applied in 0.1 ml to a column of 12 g Sephadex LH-20 in methylene chloride/benzene 2:l (column dimensions: of 1.0 ml were col0.85 x 49 cm). Fractions lected between 0.6 and 1.0 TBV. Each fraction containing radioactive material was taken to dryness at room temperature under a stream of nitrogen. A fraction containing pure cholesterol (according to TLC and GLC) was quantitatively analyzed by GLC of the TMSi derivative. By measuring the radioactivity in the same fraction, the specific activity of the cholesterol could be determined. By dividing the total amount of radioactivity found in the original chloreform/methanol 1:l extract with the specific activity of the cholesterol recovered in the solvolyzed sulfate fraction, the total amount of cholesterol excreted could be determined. RESULTS Purification methanol be

logical

the

cholesteryl

extraction

a very

nary

of

of

efficient

material

way

of

preceeding

section

14 C sulfate

from

the

four

mine

if

cholesteryl labeled

1000 The

of

steryl

that

0.2M

ammonium

thus

sulfate

would

in

not

was

was

been

lipids

from

for

of

added

with

To

present,

detected.

the

after

aqueous

according 0.3"mg On

chole-

the

deter-

bound added chloro-

extracted

in 75%

in

extracts

specifically

remaining

bio-

prelimi-

86-10456)~

miscible

to

described

fecal

purified

1. If

known

recovery

contfnously

carbonate

is

procedure

(range:

residue

fig. have

98%

chloroform/

used

preliminary

was

obtained

outlined

the

contained

a fecal

extraction

material

procedure

was

was

average

the

material

sulfate

compound,

ml

in

total

solvent

With

the

subjects

fecal

form/methanol

this

Because material

to extract

feces.

steryl-4-

the

lyophilized

(IT),

extraction

sulfate.

with ethanol. to

the

of

chole-

basis

of

STEROIDS

194

this

the

finding,

sidered

neutral

pyridine

applied

to

the

sulfate

by

terial

successive

in

sulfate

determining

by the

the

24.8~43,8$).

was

con-

of

lipid

It

can

be

calculated

on

the

average,

1.4%

is recovered

of

in

sulfate

was

the

the

the

been

final

pyridine

was

the reco-

34.4%

(range:

the

summarized

of

in

a weight

in

cholesteryl

ba-

subsequent in Table

the

the

was ma-

On

in

figures

weight

the

described

chloroform.

has

(18)

C-labeled

of

of purification

from

14

of

extracts

material

procedures

previously

hormones

conditions

with

Th e degree

chromatographic

steroid

99% of ch 0 1 esteryl

extractions

recovery

method

recovery

chloroform

section, two

of

the

Under

solution.

preceeding

tract

procedure

extraction

microanalysis

evaluated

sis,

extraction

satisfactory.

The

vered

11:2

table

1.

that,

chloroform

ex-

sulfate

frac-

tion. Table CHROMATOGRAPHIC

1.

PURIFICATION

OF

Mean Sephadex G-25-36 CHC13/CH30H 1: 4

6.8

7805

62.3

-

96.4

9

11.7

3.9

-

26.3

10

chromatograms

are

shown

-24.7

18

in

Representative

radioactive

of experiments

15.1

CM-Sephadex-LH-20-NH4+ in CHC13/CH30H 4:l

pooling

No,

Range

in

Amberlyst-15-NH4+ CHC13/CH30H 1: 4

the

SULFATE

Weight of fraction as per cent of total material applied to the column.

Procedure

where

CHOLESTERYL

of

material

labeled

fractions

is lost

in

each

is

in

fig.

indicated.

chromatographic

2, Some step.

Feb. 1968

This 80s

STEROIDS

in part of

explains

the 14

cholesteryl-4-

net

195

recovery

C sulfate

in

of

the

approximately

final

fraction.

-mg

cpm.164 (=-eC)

(=-_) -

cpm.lci4

mg (:-)

(z-e-)

i

80 -

I

-

t I’ I’ -

: ’

10 I

60

40

20 -

‘I ‘I ’ 1 ’ 1

30

-

’ ’ ’ ’

20

15 -

1 1 1 1 10

’ 1

-

5-

10 5-

20

I 20

0

40

ml effluent

60

80

ml effluenl

purification of 526 mg of Fig. 2. Left: Chromatographic a chloro% extract applied to 130 g of Sephadex G-25-36 in CHCl /CH OH 1:4. Column dimensions: 3.1 x 62 cm. Flow pooled as indicated by bars. rate O. 8 ml 3 min. Fractions Right: Chromatographic purification of a fraction from a different analysis, which has been pooled similarly to that shown in the left chromatogram and eluted from an Amberlyst-15-NH4+ column (see fig. 1). Column: 8 g of CM-Sephadex-LH-20-NH4+. Column dimension: 0.9 x 49 cm. Solvent: CHC13/CH30H Fractions 4:l. Flow rate 0.8 ml/mine pooled as indicated by bars. Identification part

(40

Pg)

of of

the

CM-Sephadex-LH-20 spot

when

solvent

pooled

column

analyzed system

cholesteryl

for

methanol/chloroform

by

sulfate.

radioactive (cf.

fig,

yielded

sulfates. small

analysis

material

2)

a two-dimensional

steryl

TLC

revealed

from onl,

development

Q:ystallization somewhat

of

fluffy

a

+' one in

the

from needles

196

after

which,

drying

melting

point

steryl

sulfate

(7)).

camp. between

182-187'

of

the

sulfate

sterol

cal

cholesteryl

(first third

ammonium

of

vealed

two

10

behind on &F-l

the

cm,

The

The

main

and

lesteryl-TlWi.

SE-30

Since

the

and

after

second

de-

agreement

authentic

on TLC

GLC peak

of

using

with had

the

with

repete-

7:l cm and acid

the

mobility

the

same

was

TMSi

activity

10

fe-

sulfuric

compound of

the

acetate

development:

component

specific

185'

solvolysis

Charring

one

chole-

3).

(Fig.

analyzed

spot.

showed

feces

a

ammonium salts of synthethe compound isolated from

contaminating

cholesterol

and

the

perfect

in benzene/ethyl

18 cm).

spots.

cholesterol.

was

plate

development: development:

the and

isolated

the

from salt

spectra of sulfate (A)

sulfate

showed

had

for

(6)

197-201°

isolated

component

elution

salt:

temperature

(Reported

spectroscopy

substance

The

at room

decomp.

ammonium

Fig. 3. Infrared tic cholesteryl feces (B).

tive

in vacua

Infrared

cholesteryl

as

11:2

STEROIDS

found

re-

just

derivatives

same

RRT

as

determination

cho-

Feb. 1968

of

STEROIDS

the

cholesterol

ed upon

quantitative

minating

compound

sterol

portion

phadex

LH-20

the

liberated

had

from gave

contaminant.

compound

but

GLC

of

to be

the

later

from its

the

TMSi

removed.

solvolyzed

a partial Early

197

sulfate

fraction

derivative

the

Chromatography sulfate

separation

cholesterol

fractions

of

pure

contaof

fraction

the

on Se-

cholesterol

fractions

were

rest-

and

contained

(Fig.

the

4).

Fig. 4. Thin-layer chromatogram on silica gel of two sterol fractions obtained after fractionation of solvolyzed sterol sulfates on a Sephadex LH-20 column (see text). mixture of 5a-cholestan-3P-ol and 5cL-chole1. Reference Stan-3cl-01 (upper spot). fraction from the Sephadex LH-20 "Early11 cholesterol 2. column. fraction from the Sephadex LH-20 3. "Late" cholesterol colLlmno 4. Reference cholesterol. Solvent system: benzene/ethyl acetate 7: 1 used in three consecutive developments (first: 10 cm; second: 10 cm; third: 18 cm). These

latter

minations. of

fractions GC-MS

cholesterol

TMSi

derivative

lesteryl

TMSi.

were

verified since was

the

used

the mass

identical

for

TLC

and

quantitative GLC

spectrum with

that

identification

obtained of

deter-

of

authentic

the cho-

11:2

STEROIDS

198

Quantitative

studies.

ted

2. From

in Table

ween

the

a value be

for

from

steryl

Since

the

fraction

amount were

tion

than

fraction

in

impurities

turbed

the

in

of

the

ed by

the

these

experiments

on

the

recovery

lipophilic

the

two

values

in Table

rences

in

found.

However,

the

4,

it

pattern in

the

all

sulfate

compounds

are

obtained

of

in the

some

fracpresence

extent

dis-

ketones. is

illustrat-

3.

in Table instead

of

eluted

The

In

copro-

together

(19).

data

in Table

is

seen

of

cholesterol

that

wide

2 and

from

individual

metabolites

the

subjects,

excreted

found,

Similarly,

procedure

used

could

calculations

steroid

was

columns

quantitative

to

summarized

cholestanone

the

bet-

N-sap. N

determined

which of

and

values

because

chromatographic

Sephadex

the

cholesterol

fraction

the

excreted.

mainly

N,

N)

occasionally

1) in

accurately

presen-

content

fraction

were

fig.

experiments

However,

From

N

of

to use

GLC-quantifications

reliability

stanone.

values

are

sterol

(fraction

content

(see

more

results in

of metabolites

ketones

of

extract

accurate

steroid SK

difference

ester

more

SE-sap.

total

quantitative

negative

considered

the

the

petroleum

the

obtained.

it was

of

light

The

varied

relative within

the diffe-

were amounts

narrow

of

limits.

DISCUSSION Isolations. inert used.

To

lipophilic

ensure

good

Sephadex

yields gels

of

have

cholesteryl been

sulfate,

systematically

d

III

Subject

6.3

Weight% of total lipid 1.0

3.0

58.3

(x4.4;’

see

$

**

l

Sumber

Fig.1.

j

Table

(1)

1

AND

d

IV

9

II

iubjec

2

(4

in fraction

S are not

1.3

3.7

142

23.0 (4) ,(20.7-24.2)

(;;::-g!5)

5.3

16.8

357

(:::;$'8,

5-en-3/3-01

metabolite

EIETABOLITES

of cholestane.

4.9

14.2

547

(;:68-%,

441 (2 (410-471 I

1.7

5.3

113

(1.7-2.5

2.1

115 (2 (111-118

111 (3 (110-111

5P-3P-01

:holesterol

CHOLESTEROL

ketones

features

(W

and the steroid

structural

1.3

2.9

255

(z-26;

325 -

0.8

2.4

46.5

5p3-one

determined

OF CHOLESTEROL

in parenthesis.

N-sap.

to specific

in fraction

of experiments

The sterols

All notations

refer

designation

E

l

Fraction

16.5

l

1.4

37.9

Weigh+ of total liviri

3.2

3280

Yeight$ of fraction N

276

(:i%lia

Total

sulfate

Solvolyzed

SK

SIC-sap.

N-sap.

i

5-en-3p-01

metabolite

EXCRETION

($::-4~50)

18.6

N

364

Weigh& of fraction N

(R:kb:‘l,

362 (2 (359-364

Total***

Solvolyzed sulfate

SK

W-SOP.

S-.¶Clp.

306 (2 (288-324

5p-3p-01**

holester

FECAL

(4

included.

1.9

5.7

219

I

I

(me)

(1)

(:;:-2$3

(:;L;:

0.1

0.3

5.3

5.3 (3.5-7.0

22.6

5p-3-one

determined

0.1

0.3

12.2

(10.3-14.:

12.2

0.2

0.6

11.8

(;:,:I;‘?,

46.8

(4)

(1)

4-en-3-one

200

STEROIDS

11:2

I

I

I

k

0

A.2

UY

d

zl

Feb. 1968

STEROIDS

ch .

201

co .

.

m

2

@.I .

s

ct .

\D .

2 2

ol .

s ln

d

b

.

a3 ;

%

0

n

d t . n

.

. 7

ot

rl

d cd

o\

c; c-4

*

t

202

STEROIDS

To

separate

tition

65$

between

aqueous

since the

it

mained

is

in

added

in

traction

light

the the

-36

should

the

salts

would

presumably of

It

Sephadex

the

LH-20 thus

is

This

due

has

to

the

the

on

been In

necessary

ion

to of

been

ensteryl

Sephadex

radioactive

difference

peak.

present,

G-25If they

different

elution on

partition in

from

volumes

lipophilic

(2).

It

is

a homogenous

achieved

the

was

in

sulfates

sulfate

form

re-

ex-

salt

volumes

steryl

the

salt

(20).

the

(20).

salt

by having

present

study

there-

a salt

CM-Sephadex

used

as

a stationary

excess

of

ammonium

of

the

fecal

chole-

data:

The

chromato-

ions

phase, in

system. The

rests

properties

elution

of

with

excess

Subsequent

had

chosen

pyridinium

pyridinium

one

a liquid-gel

ammonium

sulfate

only

salt

of

sufficient

sulfate

had

of

in

was

together

amounts

amount

phase.

previously

Identifications.

compound

the

salts

chromatographic

graphic

of

that

phase

providing

steryl

yield

to keep

mobile in

in

pyridinium

important

form.

be

have

carbonate

sulfate-chloroform

thus

cholesteryl

different

The

a par-

carbonate

removed

minor

pyridine

yielded

is believed

between

fore

of

the

only

chloroform

chromatography

that

way

be

lipids,

ammonium

Ammonium

could

phase.

fecal

and

used.

subsequent

other

the

this

a quantitative in

neutral

and

aqueous

step

sulfates

in

was

volatile In

and

petroleum

ethanol

ethanol.

sure

acidic

11:2

identification on

were

liquid-gel

the

following

the

same

as

those

chromatography

and

of

the

TLC.

synthetic

Treatment

Feb. 1968

with

dioxane

The

infrared

cal

(fig.

1365

The

of

with

strong

at

of

lished.

an

and

product

that

the

In

of

the

subjects

revealed

proportion

was

surprisingly

tively

any

cal

in human

cm

-1

sterol

The

and

strong

indicate

the

liberated

solvolysis however,

is now

was

not

suggest

being

from

estab-

that

it

out

to

carried

not

have

seemed

and

is

found

sterol

to be

sulfate

is of

(6).

to

the

it

from

endogenous of

four

to

ester the

Cholesteryl a quantita-

3p-Hydroxy-5/3the

appears

isolation

sulfate

present.

constitute

the

patterns,

correlated

derivative.

absent

and

the

not

as

habits.

from

excretion

excre-

favourable

dietary

fractions

excreted was

chole-

important

same

derivative

fraction,

by

or not

considered

individual

not

fecal

the

steroid

sterol

was

whether

it was

cholesterol

other

supported bile

1440

a quantitatively

constant

important

sulfate

be

neutral

of

cholesteryl

further

at

(21-23).

1200

(22,23).

data,

highly

however,

-cholestanols steryl

peak

upon

Work

did

fecal

the

sulfate,

around

cholesterol,

subjects

of

bands

investigating

could

Although

amount

absorption esters

identi-

(24).

sulfate

tion

4).

by GC-MS.

were

contaminating

GLC

confirmed salts

salt

sulfates

Quantifications. steryl

the

(fig.

this

the

as

ammonium

sulfate

and

of

TLC

cholestanol

the

ammonium

steryl

The

confirm

for

3200

nature

fecal

cholesterol

spectra

typical

band

presence

liberated

3),

cm -I

broad

the

203

STEROIDS

solvolyzed

possible

that

fe-

origin.

This

is

cholesteryl

sulfate

204

STEROIDS

In

a study

a half-life fate

and

of

of

two

a production Furthermore,

half

the

sterol

and

a daily steryl 60

mg

lesteryl the

rate

cholesteryl

from

apparent

Gurpide

et

pathway

for

al. the

35

was

plasma

and

163

showed

acid

plasma

The

et

converted esters.

of

respectively

contained

sulfate.

for

authors

fatty

cholesterol).

study

of

produced

sulfate/day,

in our

if

amount

Gurpide

hours

these

elimination

of

15

14 and

tively. of

subjects,

17 and

al.

(25)

cholesteryl mg/day,

14.4-84.7

portions mg

of

that

at

into

free

chole-

This

would

mean

80

mg

of

cholesterol

studies

are

needed

to

agreement

between

our

values

and

that

elimination

fecal of

excretion

serum

least

of

chole-

to

13 and

feces

Further

indicate

sul-

respec-

(corresponding

daily

found

is

cholesteryl

analyzed as

cho-

establish those

of

a major sulfate.

ACKNOWLEDGEMENTS We are very much indebted to Dr. J. Sjovall for facilities put at our disposal. The skilful technical assistance of Miss Kerstin Persson and Miss Christina Larsen is gratefully acknowledged. This work was supported by grants from Karolinska Institutets Reservationsanslag, Sallskapet for Medicinsk Forskning and the Swedish Medical Research Council (Grant No. 13-X-219 to Dr. J, Sjovall). REFERENCES 1. 2.

3. 4. 5. 6.

Moser, H.W., Moser, A.B., and Orr, J.C., BIOCHIM. BIOPHYS. ACTA 116, 146 (1966). Sjijvall, J., Haahti, E., and Nystrom, E,, ADVANCES IN CHROMATOGRAPHY Eds. Giddings, J.C. and Keller, R.A., Marcel Dekker, Inc., New York, N.Yo, 1967, in press OF PRACTICAL ORGANIC CHEMISTRY, Vogel, A,J., A TEXTBOOK Longmans London, 1964. Pamphlet, Waters Associates, "Porapak 811 Advertising Mass., USA. Inc., Framingham, Kornel, L., Kleber, J.W., and Conine, J,W., STEROIDS 4, 67 (1964). and Lieberman, S., BIOCHEM. BIOPHYS. RES. Drayer, N.M., COMMUN., IS, 126 (1965).

Feb. 1968

7.

Mandel,

8.

McKenna,

(1915)

J.R.,

and

Neuberg,

C.,

J.,

and

Norymberski,

11. 12.

13. 14. 15. 16. 17.

18.

Bray, G.A., ANAL. BIOCHEM. Nystrom, E., and Sjovall,

(1965)

BIOCHEM.

Z. 71,

186

* J.K.

(1957). 9. 10.

205

STEROIDS

1, J.,

J.

CHEM.

SQC.

274 (1960). ANAL. BIOCHEM.

3889

l2,

235

o

Nystrom, E., to be published. Wusteman, F.S., Dodgson, A.G,, Lloyd, A.G., Rose, F-A., and Tudball, N., J. CHROMATOG. 16, 334 (1964). Freeman, C.Pe, and West, Da, J. LIPID RES. '& 234 (1966). van Dam, M.J.D., De Kleuver, G.J., and De Heus, J.G., 4, 26 (196o)e J. CHROMATOG. Eneroth, P., Gordon, B., Ryhage, R. and Sjijvall, J., J. LIPID RES. 2, 511 (1966). Eneroth, P., and Nystrom, E., to be published. Pedersen, T.A., ACTA CHEM. SCAND. 16, 374 (1962). McKenna, J., and Rippon, A-E., BIOCHEM. J. a, 107

(1965). 19. 20. 21. 22. 23. 24. 25.

Eneroth, P. and Nystrom, &, 149 (1967) Sjovall, J., and Vihko,

E., R.,

BIOCHIM. ACTA

CHEM.

BIOPHYS. SCAND.

ACTA 20,

1419

(1966). R., Vincent, V., Caltop, J., as cited by Dixon, Kase, N., STEROIDS, 6, 757 (1965). ABSORBTION SPECTROSCOPY, Nakanishi, K., INFRARED USA 1964. -Day, Inc., San Francisco, Roberts, K.D., Bandi, L., Calvin, H.I., Dricker, and Lieberman, S., BIOCHEMISTRY 3 -2 1983 (1964). Eneroth, P. and Nystrom, E., to be published. Gurpide, E., Roberts, K.D., Welch, M.T., Bandy, and Lieberman, S., BIOCHEMISTRY 2, 3352 (1966).

and HoldenW.D.,

L l

’