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Saturated metabolites op progesterone in human myometrium during pregnancy
65 SATURATED
METABOLITES
IN HUMAN
OF PROGESTERONE
MYOMETRIUM
DURING
PREGNANCY
by Hiakan
H,
I. Frauenklinik Univereitlt
und
Hebammenschule
Munchen,
D - 8 Winchen
11,
Maiatral3e
Germany
Received: 10/l/75 SUMMARY A method for the aeparation and quantitative determination of epimoria 3q/D-hydroxy-54/&pregnan-2O-onea bygaa liquid chromatography and elootron capture detection ia preaented. Reliability of the method and applicability to biological material waa teated.In one total human uterus and 20 aamplea of myometrium the concentration of epimeria pregnanolonee waa determined. 3D-hydroxy-Sa-pregnan-20-one and 3a-hydroxy-5ty-pregnan-20-one were preaent in similar quantitative range aa progesterone and 20a-hydroxy-&pregen3-one.A correlation between progesterone and concentration of metabolitea could not be eatabliahed. 3B-hydrory-5I3pregnan-20-one wae not deteated in the tissue.
INTRODUCTION In vitro muaale
from
ltudiea
rat
ia metabolized and
alao
POa-OH-P
in human (5)
myometrium
there
Therefore the amount
lee of human
Vo'owne27, Nmber
uterine
Z
during
from
(PO&-OH-P)
on progesterone pregnancy
ia no information
of metabolitea
in uterine
(4) progesterone
to 20Whydroxy-4-pregnen-y-one
of the report
tiaaue.
that
(3) and human
lxaeption
& Zander
determine
(1,2),aheep
producta.
centration thie
demonstrated
Sa-aaturated
With
baum
have
endogenoua
a aeneitive of 5a-saturated
method
and
by Runne-
about
the con-
progesterone waa
devised
metabolitee
in to
in camp-
muscle.
m
WBaOXDI
January,
1976
66
S
YJ?=EIEOXD6
Solvent% were redistilled before use. Reference steroids were obtained from commercial sources and recryF stallized. Thin layer chromatonraphv (TLC) was performed on 20 x 20 cm glas% plates coated with silica gel GF 254 (Merck AG) at 0.25 mm thiokneas. After drying at roomtemperature the plates were activated at 110 C and stored in a desiccator until used. Steroids were located by spraying with anisaldehyde-sulfuric acid and heating at 100 C for 10 min. Radioactive labelled material was detected using a thin layer scanner (Berthold & Frieseke, Mod. LB 2723). Gas liauid chromatography (GLC): A Packard GLC appa(Series 7400) equipped with two flame ionisation deID ) or two electron capture detectors (ECD) was foil was the electron emitting source in the ECDs. Gla%s columns of 3 ft. and 6 ft. length and 4 or 2.5 mm internal diameter were prepared azdpacked as described (6) with Gas Chrom Q (100 - 120 me%h, Serva AG, Heidelberg). The following istationary phases were used: QF-1 (15 and 3$)! XE-60 (2% and 3%)~ OV-225 (396). Nitrogen purified by microsieve filter% was used as carrier gas. Columns were maintained at 240°C and detector% at 270°C. Testosterone or testosterone monochloracetate was used as internal standard during GLC. ratus
Trimethylsilyl (TMSI)-ethers were preDerivatives: ust prior to GLC as de%crZbed (7). The reagent was N-methyl-N-trimethylsilyl-tri~oroacetamida,M~cherey & Nagel Comp., Dtiren). Steroid monochloroacetates (ClAc) were formed a% described (8) Hexadecafluoronanoyl derivatives (HFN) were prepared according to the procedure of Kirschner and Taylor (9). Sulfate derivative% of steroid% were prepared as described previously (10). All derivative% with exception of TMSI-ethers were recrystaLlized before u%e. Tissue aamplesr Two specimens of myometrium of the pbstovulatory &a:;do4f2~;Ycle, 18 specimen* from 9 uteri between the 11 week of pregnancy and one total uterus were obtained at hysterectomy or interruption of pregnancy for medical reason%. The tissue was blotted, minced an homogenized with an Ultra-Turrax after adding two volume% of digtilled water. Extraction: After adgition of (3H)-3D-hydroxy-s-pregnan-20-one (3G-%P) and ( H)-k-sulfoxy-5D-pregnan-20-one of both 50.3 Ci/mmole, (3%+5B-Pso~),( specific activity New Englan Nuclear),the homogenates were extracted 4 times with 20 ml of diethylether-ethanol (1:3, vfv).The extracts were evaporated in vacua and the residue dissolved
S
67
lC3EOXDll
in 10 ml of 70% methanol which was stored at -2OoC overlipid material was sedimented and night. By centrifugation the supernatant extracted with 3 x 10 ml diethylether resulting in the "free" fraction.The aqueous phase containing the "sulfate" fraction was extracted with 3 x 10 ml n-butanol. Chromatoarauhvt The butanol extract was evaporated and chromatographed on a 1 x 15 cm Sephadex-LH-PC column in system chloroform-methanol lrl, containing 0.1 mM NaCl (11). The purified "sulfate" fraction obtained from this chromatography was subjected to solvolysis as described (12). The solvolysed and the "free" fraction were chromatographed on TLC in system chloroform-acetone 9rl. The monohydroxy-pregnanes were eluted as a group as they migrated with Rf-values of 0.52-0.57 in this system. The eluted fractions were further chromatographed in system n-hexane-ethyfaoetate 3:l with three developments.In this step J&-hydroxy-5D-pregnan-20-one (%-50-P)was separated from the other epimers.After derivative formation (Monochloroacetate)the fractions were purified by another chromatography on TLC in system benzene-ethylacetate lO:l.The solvent systems and Rf-values are listed in table 1. Quantitative determinationrAfter removal of an aliquot to determine procedural losses by counting the remaining radioactivity the fractions were subjected to GLC and electron capture detection. Testosterone-monochloroacetate was used as internal standard and the mass of the individual steroids was calculated according to the following formulas P (ng) = R x SF x PF x IS x MP P
= Pregnanolone
R
= Recovery Peak area = Peak area
SF
Peak Peak
area area
of internal of standard
of oreunanolone in sample of internal standard in sample
PF
=
IS
= ng of internal
MF
=
Molecular Molecular
standard pregnanolone
standard
weight weight
added
to sample
of nreananolone of pregnanolone-monochloroacetate
20a-hydroxy-4-pregnen-3-one (2QcyJOH-P) was determined by GLC as described (8), Progesterone in tissue extracts was determined by radioimmunoassay (13) using an antiserum against lli3-OH-progesterone-hemisuccinate serum albumin. The extracts were purified before the binding assay by chromatography on Sephadex LH-20.All determinations were performedin duplicata
S
68
TBEOlCDI
Solvent I
Compound
systems
II+
III
IV
Progesterone
.80
.43
2orr-OH-P
.53
.27
POD-OH-P
.52
.29
3tY-5ar-P
.55
.49
.69
l34
3a-_5a-P
.54
.42
.70
433
30!-5a-P
.57 l57
935
3a-go-P
l54
.68 .66
l39 .26
3a-A5-P +
.56
.47
.65
.36
200
5&-p**
.61
.46
SQI-P-dione 5D-P-dione
.86 .86
.70 l66
Testosterone
.46
023
.30
.68
Rf-values, Table 1: Thin layer- chromatography, * = 3%hydroxy-5-pregnen-ZO-onej + e three developmentsj ** = 20Q-hydroxy-5Q-pregnan-3-onej I = Chloroform-Acetone 3rtj III = Benzene-Ethyl9rt; IL I n-Hexane-Ethylacetate 8:2. acetate 10:lj IV = n-Hexane-Diethylether
RJEWLTS 1.
Recovery From
taining labelLed cessed.
and
accuracy;
a pool
of Krebs-Ringer
3&%-P,
each
of 5a-P-3a-so4
and
labelled
compounds.
labelled
compound
mination
by GLC
recovery,
and
5a-P-3%SO4
compounds, 5 ml
bicarbonate
12 samples contained 3600
yielded
representing
ng 3&5ar-P,
cpm reap.
40.1 were
the corresponding
of 5 ml were
780
2900
f. 6.5% of the
recovered. 746 f 96% 3
buffer
The
taken the
contritium
and pro-
same
amount
cpm of radioactive "free"
radioactive
quantitative
105 ng,corrected 14% of added
by
3&-!$a-P.
detertracer
The
recovery 11s.
56,8$&
94% 2 8% of the ments lues for
with
of radioactive
added
biological
of recovered the
722.9
By GLC
"free"
f
labelled
56 ng were
5iacP-30-SOb
measured
pregnanolone-sulfate. material
radioactive
compounds
and
(Exp.
is
In the experi-
1-21,
material
was
which
table
were
31% f. 6% for
4) the va-
51% +
10.5%
the "sulfates".
2. Precision: The mated
precision
from
3&w-P
the duplicate
and
5&-P-32-SO4
variation
was
pound
7.896 for
and
of quantitative
1496 for
determination
experiments
(n P 12) with
to EBB-buffer.
The
the determination
the determination
was
esti-
added
coefficient
of the
of the
free
of
com-
sulfate.
7. Sensivitv: As
5 ng of the monochloroacetyl
quantitated of the method determinations
sufficiently is about are
by GLC 50 ng
derivative
the practical
steroid
per
can be sensitivity
sample
if several
to be performed.
Fia.1: Gas-liquid chromatography of monochloroacetate derivatives of pregnanolones isolated from myometrium (Exp.1). Column: 3$ QF-1, 6 ft. On the absaissa time in minutes. For details of GLC see methods. The shaded areas depict the amount of authentic substances injected together with the biological sample. 1 = P-hydroxy-SQ-pregnan-20-one; 3 I 3S-hydroxy-5-pregnen-20-one; 4 = 3B-hydroxy-%-pregnan-20(Internal standard). one; 5 = testosterone-ClAc
S
70
lclpEOSDI
4. Specificity The ral
specificity
of
chromatographic
compounds
with
monstrated measured
the method
steps;
two of
the epimeric
compounds
with
authentic
peak
sample
to GLC
was
(Figl).The
subjected separation
in table
2 where
spect
to testosterone
Table
2: Relative
relative
time
of
when
time
1.0
1.0
1.0
1.0
200r-OH-P
1.87
1.54
1.53
-
2Of3-OH-P
1.71
1.36
1.40
-
zau-W-P
1.25
-
-
-
was
de-
of the
demonstra-
the biological
with
the
standard
pregnanolones retention standard
is demon-
times are
(Rt) during
testosterone
Testosteron,
was
seve-
tracer
identity
material
together
as internal
retention
(Retention
The
increase
of epimeric
by
of added
pregnanolones
by radiogaschromatography.
ted by a symmetrical
strated
is determined
the identity
with
re-
compiled.
GLC
= 1)
1.0
1.0
1.0
1.84
1.70
1.56
-
-
1.59
1.42
-
-
-
-
-
957
.55
.5?
1.22
1.15
1.0
1.0
%-W-P
.61
957
.59
029
.58
.52
3n-5o-P
.59
.5?
.59
.30
.59
.55
.5?
.54
.5?
3lz-513-P
-68
.58
.61
-66
.5?
-57
.85
.68
.?4
.81
.?2
.58 l?2
.61
3a-5g-P
.52 .34
.go
.?l
3g-AK43
.84
‘68
.?l
.48
.?8
.65
.66
.80
l66
71
This ment
1 by measuring
rivativea ment
criterion
one
total
uteru8
columns (960
purified
of the indicated
retention strated
time under
completed
GLC.
g wet weight)
as the authentic
different In this
was
are
with
standarda
conditions
de-
experi-
extracted
same
could
and
In
compiled,
the
The
relative
be demon-
of GLC.
n&g
Compound
Table
in experi-
chromatography.
experiment
compounde
six different
using
during
by additional
2 the rerrulte of this
presence
and
the 8p111efractions
and different
the extracts table
W~IB checked
3R-5u-P
+
+
+
+
+
4
18.7
h-&-P-S04
+
+
+
+
+
+
35.6
3&5&P
_
-
-
-
_
-
-
3S_5S_P_SO4
_
-
-
-
-
_
.
32..ggl-P
+
+
+
+
+
+
38.7
3a-5cr-P-SO4
+
+
+
+
+
+
9.4
3a-5a_p
_
_
_
_
*
-
-
3nL5%P-SO4
*
+
+
+
+
+
15.2
3l3-A5-P
+
+
+
+
+
+
25.8
313-A5-P-SO4
+
+
+
+
+
+
a.9
3:
Exo.1: Isolation of Pregnanolones from myometrium (970 g + indicate8 demonstration tiabue, 40. week of pregnancy). of compound with identical relative retention time ae the reference compound (these values are shown in table 2) a symmetrical peak increase after injection of authentic compound8 together with analysis sample.
S
72
Concentration
of metabolites
In experiment could
from
(k-%-P)
(3&5c+P), fate,
and
and
addition
3)
the
three
tissue.
tissue:
epimeric
as
the
free
compound
3a-sulfoxy-50-pregnan-20-one there
its
sulfate.
not
be
was
The
found.
compounds
a-hydroxy-W-pregnan-
3B-hydroxy-w-pregnan-20-one
present
both
in uterine
1 (table
be isolated
20-one
TREOXDI
and
(50-P-3&-S04).
3D-hydroxy-5-pregnan-20-one
epimer
30-5o-P
with
was
as sul-
metabolite
and could
3Lb5l3
the configuration
the main
In
(d5-P)
in this
uterus. In experiments ted. the
small
samples
each
same
uterus.
In table
oompiled. 35-k-P
The and
centration only was
2-21
Two
from not
In a water
blank
detected
titative (Exp.
of are
compounds
samples
were
the
processed
by
which
in
which
In the other
determination
extrac-
parts
results
or below
peaks
were
different
4 the quantitative
sulfoconjugate
detected
samples
from
to 296 rig/g tissue.
6 samples.
were
tissue
taken
predominant
its up
were
these
were
present
3a-5a-P
was
specimens
sensitivity
this of
the described
could
interfere
of the different
in con-
isolated steroid
the method. method
with
no
the quan-
pregnanolones
22). Progesterone
determined
also.
concentrations the reported The
could between
The
range
results
one
tissue
included were
the nonpregnant
amount of
be detected,
samples
in table
found
in the
uterus
of progesterone
the saturated There
the concentration
metabolites
are
steroids
these
were
4. The
to be within
(5).
from
a small nor
~OCY-OH-P in
of these
tissue
contained 2OeOH-P
and
was
no
but
2 and
3)
neither
progesterone statistical
of progesterone
tissue.
(Exp.
metabolites
correlation and
those
of
the
poetov. Ph. postov.Ph.
IO. 10.
Il.
2 3
4 5
6 7
18. 18.
18. 18.
18. 18.
24. 24.
41. 41.
12
13
14 15
‘16 17
18 19
20 21
22
Water
12. 12.
11
IO
5ml
5::
;::
;::
;:::
5.4 5.2 I
;::
-
28.1 17.5
23.8 36.0
114.4 75.9
121.1 116.9
106.7 81.2
%*43.
135.6 90:2
;::
11.
11.
x
zx
z
40.4 18.9
5.2 5.3
55.8 11.5
11.7
6.9 12.2
20.7 23.9
Z:;
37.2 20.4
7.8 11.7
2:: 245
I
52.7 40.6 50.6 81.8
10.0 10.5
120.9 33.1
10.0
35.2 10.0
10.0 110.8
72.5 30.3
9.4
22.6
18.6 12.4
18.7
128.5 110.6
I
5ty-P-3f3-so4
Wet weight)
10.0
32.4 30.4
32.3 38.0
21.2 61.4
23.4 52.9
35.0 41.9
249.1 296.4
10.0
22.5
24.6 19.2
38.7
(na;/g
3u-5a-P
muSCle
3i3-5qs-P
uterine
20 Cy-OH-P
in
I de teranined ;:;
not
4.9 5.2
970.0
Prog.
and metabolites
11.
blank
40.
1
Tissue (g)
4% Progesterone L
Pxp. Week of Pregnancy lo.
Pablo
24.0 13.3
12,9
IO
35.6
5~P-srrso4
DISCUSSION Assuming organ
that metabolism
is related
to hormone
of a hormone action
gained
by determining
the concentration
parent
steroid
tissue
From
in the
the 20 isomeric
from
progesterone
four
which
special
are
reduced
shown
to occur
ring
A was
found
target
By
tissues
these
be formed
depending
The
quantitative
ses
fundamentally
3 and
any
which
tissue may
steps
on
four
the enzyme
determination the
separation
the
molecule
5 only
the
are
which
saturation
be regarded
of has of
as so
(14).
epimeric
compounds
patternmin
of
of
be
can originate
steroid
of progesterone
metabolic
only
to 20q-reduction
in almost
target
could
the metabolites.
which
at positions
in organs
not of
of the
In contrast
interest.
also
compounds
by reduction
been
called
but
in its
information
these
may
the cells.
metabolites
of epimers
and
suppo-
sensitive
detection. Both and
criteria
electron
Adlercreutz the
tion
by GLC.
of TMSX
these
and
and
liquid
These
to about
chromatography
Chamberlain
& Thomas
SjZSvall (17) have
the quantitative
derivatives
methods
by gas
detection. (16)
et al.
separation
nanolones
are met
capture
authors
100 ng
reported
determination
however
which
limits steroid
used the
(15), on
of preg-
flame
ionisa-
sensitivity
in biological
of mate-
rial. Concerning nes
and
tion Molen
electron
separation
fs available. & Groen
progesterone
nanolones. these
(8) for
As
it appears
detection by GLC
the quantitative
electron
by
that
capture
the relative
as compared the
of pregnanololittle
to the method
smaller
of van
derivative detection
retention
to TMSI
informa-
determination
the monochloroacetyl
and
shown
derivatives
columns
In analogy
we used
gaschromatography
capture
of derivatives
ethers
derivatizing
of for
of preg-
times on
der
of
the same group
of
the monochloroacetate epimers sated and
during
however
longer
tection
les
to buffer
of uterine
GLC-methods This thod
by
are within
the
which
conditions
been
with
samples
of step
biological range
specificity
samp-
of other
of the me-
could
with
interfere
results
elithe
of experiment
of the method
could
with
3 derivatives
1
as the
be determined
of the method
demonstrated of human
it can be
metabolites
in the
under
on 3 different
that
were
In similar
the tissue. was
The
myometrium
found
Though phase
The
in uterine
isolated
range
concentration
comparable
determination.
and
From
these
during
preg-
3a-hydroxy-5aand in the was
iso-
as progesterone
in
of 3a-hydroxy-5a-pregnan-
to 20a-hydroxy-k-pregnen-3-one. if present
by TLC epimer
the samples
before
with
tissue
from human
of cycle
and
a8 "free** steroids
quantitative
eliminated
2-21).
3I3-hydroxy-5a-pregnan-20-one
3a-hydroxy-5D-pregnan-20-one as it was
experiment
(Exp.
in human
found
fraction.
mate-
!ja-reduced progesterone
30-hydroxy-Sa-pregnan-20-one
"sulfaten
to biological
same
myometrium
stated,
are present
pregnan-20-one
tory
amount
the
phases.
has
been
of
steps
The
pregnanolones
rial
not
of de-
chromatographic
specificity
applicability
20-one
with
similar
the
the different
The
lated
for
determination.
six differnt stationary
also
compounds
epimeric
nancy.
of known
as demonstrated
true
support
results
sensitivity
(QF-1)
(17). holds
quantitative
three
phase
and precision
by addition
and
other
further
the
lr5.
muscle
provided
minating
hand
of
can be compen-
stationary
reproducibility
accuracy,
effect
a selective
On the other
columns.
as determined
raids
the separation This
chromatography. by using
ie increased
The method
diminishes
was
not
the gaschromatographic
the configuration
and
determined
to our knowledge
3ib513 has
was
never
tissue. from
contained
a uterus none
of
of the postovulathe saturated
meta-
S
76 bolites
a correlation
progesterone
or
lones
tissue
in
the
The rone could
be
between
could
from
trapped
during
side
in
tively rine
higher muscle
these
of
blood
as
and
their be
metabolites in
of
in (17)
Sjiivall
contribution
progeste-
this
tissue
cannot
on
determined
by
pregnano-
of
blood
concentrations
reported
cannot
and
originate
as
endogenous
established.
circulation The
maternal and
be
myometrium
definitively.
experiments
not
whether
answered and
concentrations
20q-hydroxy-4-pregnen-3-one
question
isolated
TmEIEOXDI
to
the
the
or
be
fetal
preliminary are
compara-
amount
in
ute-
excluded.
ACKNOWLEDGEMENTS P.
I thank Dr. Munich University, The skilful graphy. gelt is gratefully
Unterburger, I. Dept. Int. Med. of who performed the radiogaschromatotechnical asistance of Miss P. Fiinfacknowledged.
The study was supported Forschungsgemeinschaft,
by Mi
the Deutsche 109/4
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M.L.
7.
Bryson,
B., 150
Horning,
(Kbh) C.G.;
Suppl. J.
116
(1967)
REPROD.FERT.
M.J.;
AM.J.OBSTET'.GYNEC.
106,
van
E.C., GLICK
in: IX,
INTERSCIENCE
Horning, E.M., 283
(ed.),
E.C.,
Jaakonmaki, (1967)
Zander,
J.;
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(1971)
D.
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(1970)
SUPPl., 6.
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Pierrepoint,
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METHODS
PUBLISHER, Horning, P.J.,
OF
E.J.A.,
Greeck,
BIOCHEMICAL
New-York-London
M.G., Brooks,
Ikegawa, C.J.W.;
B.G.
ANALYSIS 1963
N.,
Chambaz
GASCHROM.
8.
van
der Molen, 6,
STEROIDS 9.
Kirechner, 346
10.
H.J.,
195
D., van
der Maas,
9-H-i
(1965)
M.A.,
Taylor,
J.P.;
ANALYT.
BIOCHEM.
30,
(1969)
Mickan, 477
H.,
Dixon,
R.,
Hochberg,R.B.i
STEROIDS
13,
(1969)
11.
Vihko,
R.5
12.
Jl~e,
O., Vihko,
CLIN.
Hoffmann,
14.
Morgan,
302
3781
ACTA
CHIM.
13.
4,
Grow,
ENDOCR.
ACTA
R.,
(Kbh)
23, 405
B., Kyrein,
Suppl.
SjLivall, J.,
(1966)
109
SjilVall, K.;
(1969)
H.J.,
Ender,
M.L.;
J.D.;
J. BIOL.
HORM.RES.
(1973)
M.D.,
Wilson,
CXEM.
245,
(1970)
15.
Chamberlain,
16.
Adlercreutz,
J., Thomas,
G.H.8
ANALYT.
BIOCHEM.
8,
104 (1964)
J. STEROIDS 17.
Sjiivall, K.;
H.,
Luukainen,
T., Tayler,
E.;
1, 117 (1966) ANN.
CLIN.
RES.
2, 393
(1970)
EUROP.
METABOLITES
IN HUMAN
OF PROGESTERONE
MYOMETRIUM
DURING
PREGNANCY
by Hiakan
H,
I. Frauenklinik Univereitlt
und
Hebammenschule
Munchen,
D - 8 Winchen
11,
Maiatral3e
Germany
Received: 10/l/75 SUMMARY A method for the aeparation and quantitative determination of epimoria 3q/D-hydroxy-54/&pregnan-2O-onea bygaa liquid chromatography and elootron capture detection ia preaented. Reliability of the method and applicability to biological material waa teated.In one total human uterus and 20 aamplea of myometrium the concentration of epimeria pregnanolonee waa determined. 3D-hydroxy-Sa-pregnan-20-one and 3a-hydroxy-5ty-pregnan-20-one were preaent in similar quantitative range aa progesterone and 20a-hydroxy-&pregen3-one.A correlation between progesterone and concentration of metabolitea could not be eatabliahed. 3B-hydrory-5I3pregnan-20-one wae not deteated in the tissue.
INTRODUCTION In vitro muaale
from
ltudiea
rat
ia metabolized and
alao
POa-OH-P
in human (5)
myometrium
there
Therefore the amount
lee of human
Vo'owne27, Nmber
uterine
Z
during
from
(PO&-OH-P)
on progesterone pregnancy
ia no information
of metabolitea
in uterine
(4) progesterone
to 20Whydroxy-4-pregnen-y-one
of the report
tiaaue.
that
(3) and human
lxaeption
& Zander
determine
(1,2),aheep
producta.
centration thie
demonstrated
Sa-aaturated
With
baum
have
endogenoua
a aeneitive of 5a-saturated
method
and
by Runne-
about
the con-
progesterone waa
devised
metabolitee
in to
in camp-
muscle.
m
WBaOXDI
January,
1976
66
S
YJ?=EIEOXD6
Solvent% were redistilled before use. Reference steroids were obtained from commercial sources and recryF stallized. Thin layer chromatonraphv (TLC) was performed on 20 x 20 cm glas% plates coated with silica gel GF 254 (Merck AG) at 0.25 mm thiokneas. After drying at roomtemperature the plates were activated at 110 C and stored in a desiccator until used. Steroids were located by spraying with anisaldehyde-sulfuric acid and heating at 100 C for 10 min. Radioactive labelled material was detected using a thin layer scanner (Berthold & Frieseke, Mod. LB 2723). Gas liauid chromatography (GLC): A Packard GLC appa(Series 7400) equipped with two flame ionisation deID ) or two electron capture detectors (ECD) was foil was the electron emitting source in the ECDs. Gla%s columns of 3 ft. and 6 ft. length and 4 or 2.5 mm internal diameter were prepared azdpacked as described (6) with Gas Chrom Q (100 - 120 me%h, Serva AG, Heidelberg). The following istationary phases were used: QF-1 (15 and 3$)! XE-60 (2% and 3%)~ OV-225 (396). Nitrogen purified by microsieve filter% was used as carrier gas. Columns were maintained at 240°C and detector% at 270°C. Testosterone or testosterone monochloracetate was used as internal standard during GLC. ratus
Trimethylsilyl (TMSI)-ethers were preDerivatives: ust prior to GLC as de%crZbed (7). The reagent was N-methyl-N-trimethylsilyl-tri~oroacetamida,M~cherey & Nagel Comp., Dtiren). Steroid monochloroacetates (ClAc) were formed a% described (8) Hexadecafluoronanoyl derivatives (HFN) were prepared according to the procedure of Kirschner and Taylor (9). Sulfate derivative% of steroid% were prepared as described previously (10). All derivative% with exception of TMSI-ethers were recrystaLlized before u%e. Tissue aamplesr Two specimens of myometrium of the pbstovulatory &a:;do4f2~;Ycle, 18 specimen* from 9 uteri between the 11 week of pregnancy and one total uterus were obtained at hysterectomy or interruption of pregnancy for medical reason%. The tissue was blotted, minced an homogenized with an Ultra-Turrax after adding two volume% of digtilled water. Extraction: After adgition of (3H)-3D-hydroxy-s-pregnan-20-one (3G-%P) and ( H)-k-sulfoxy-5D-pregnan-20-one of both 50.3 Ci/mmole, (3%+5B-Pso~),( specific activity New Englan Nuclear),the homogenates were extracted 4 times with 20 ml of diethylether-ethanol (1:3, vfv).The extracts were evaporated in vacua and the residue dissolved
S
67
lC3EOXDll
in 10 ml of 70% methanol which was stored at -2OoC overlipid material was sedimented and night. By centrifugation the supernatant extracted with 3 x 10 ml diethylether resulting in the "free" fraction.The aqueous phase containing the "sulfate" fraction was extracted with 3 x 10 ml n-butanol. Chromatoarauhvt The butanol extract was evaporated and chromatographed on a 1 x 15 cm Sephadex-LH-PC column in system chloroform-methanol lrl, containing 0.1 mM NaCl (11). The purified "sulfate" fraction obtained from this chromatography was subjected to solvolysis as described (12). The solvolysed and the "free" fraction were chromatographed on TLC in system chloroform-acetone 9rl. The monohydroxy-pregnanes were eluted as a group as they migrated with Rf-values of 0.52-0.57 in this system. The eluted fractions were further chromatographed in system n-hexane-ethyfaoetate 3:l with three developments.In this step J&-hydroxy-5D-pregnan-20-one (%-50-P)was separated from the other epimers.After derivative formation (Monochloroacetate)the fractions were purified by another chromatography on TLC in system benzene-ethylacetate lO:l.The solvent systems and Rf-values are listed in table 1. Quantitative determinationrAfter removal of an aliquot to determine procedural losses by counting the remaining radioactivity the fractions were subjected to GLC and electron capture detection. Testosterone-monochloroacetate was used as internal standard and the mass of the individual steroids was calculated according to the following formulas P (ng) = R x SF x PF x IS x MP P
= Pregnanolone
R
= Recovery Peak area = Peak area
SF
Peak Peak
area area
of internal of standard
of oreunanolone in sample of internal standard in sample
PF
=
IS
= ng of internal
MF
=
Molecular Molecular
standard pregnanolone
standard
weight weight
added
to sample
of nreananolone of pregnanolone-monochloroacetate
20a-hydroxy-4-pregnen-3-one (2QcyJOH-P) was determined by GLC as described (8), Progesterone in tissue extracts was determined by radioimmunoassay (13) using an antiserum against lli3-OH-progesterone-hemisuccinate serum albumin. The extracts were purified before the binding assay by chromatography on Sephadex LH-20.All determinations were performedin duplicata
S
68
TBEOlCDI
Solvent I
Compound
systems
II+
III
IV
Progesterone
.80
.43
2orr-OH-P
.53
.27
POD-OH-P
.52
.29
3tY-5ar-P
.55
.49
.69
l34
3a-_5a-P
.54
.42
.70
433
30!-5a-P
.57 l57
935
3a-go-P
l54
.68 .66
l39 .26
3a-A5-P +
.56
.47
.65
.36
200
5&-p**
.61
.46
SQI-P-dione 5D-P-dione
.86 .86
.70 l66
Testosterone
.46
023
.30
.68
Rf-values, Table 1: Thin layer- chromatography, * = 3%hydroxy-5-pregnen-ZO-onej + e three developmentsj ** = 20Q-hydroxy-5Q-pregnan-3-onej I = Chloroform-Acetone 3rtj III = Benzene-Ethyl9rt; IL I n-Hexane-Ethylacetate 8:2. acetate 10:lj IV = n-Hexane-Diethylether
RJEWLTS 1.
Recovery From
taining labelLed cessed.
and
accuracy;
a pool
of Krebs-Ringer
3&%-P,
each
of 5a-P-3a-so4
and
labelled
compounds.
labelled
compound
mination
by GLC
recovery,
and
5a-P-3%SO4
compounds, 5 ml
bicarbonate
12 samples contained 3600
yielded
representing
ng 3&5ar-P,
cpm reap.
40.1 were
the corresponding
of 5 ml were
780
2900
f. 6.5% of the
recovered. 746 f 96% 3
buffer
The
taken the
contritium
and pro-
same
amount
cpm of radioactive "free"
radioactive
quantitative
105 ng,corrected 14% of added
by
3&-!$a-P.
detertracer
The
recovery 11s.
56,8$&
94% 2 8% of the ments lues for
with
of radioactive
added
biological
of recovered the
722.9
By GLC
"free"
f
labelled
56 ng were
5iacP-30-SOb
measured
pregnanolone-sulfate. material
radioactive
compounds
and
(Exp.
is
In the experi-
1-21,
material
was
which
table
were
31% f. 6% for
4) the va-
51% +
10.5%
the "sulfates".
2. Precision: The mated
precision
from
3&w-P
the duplicate
and
5&-P-32-SO4
variation
was
pound
7.896 for
and
of quantitative
1496 for
determination
experiments
(n P 12) with
to EBB-buffer.
The
the determination
the determination
was
esti-
added
coefficient
of the
of the
free
of
com-
sulfate.
7. Sensivitv: As
5 ng of the monochloroacetyl
quantitated of the method determinations
sufficiently is about are
by GLC 50 ng
derivative
the practical
steroid
per
can be sensitivity
sample
if several
to be performed.
Fia.1: Gas-liquid chromatography of monochloroacetate derivatives of pregnanolones isolated from myometrium (Exp.1). Column: 3$ QF-1, 6 ft. On the absaissa time in minutes. For details of GLC see methods. The shaded areas depict the amount of authentic substances injected together with the biological sample. 1 = P-hydroxy-SQ-pregnan-20-one; 3 I 3S-hydroxy-5-pregnen-20-one; 4 = 3B-hydroxy-%-pregnan-20(Internal standard). one; 5 = testosterone-ClAc
S
70
lclpEOSDI
4. Specificity The ral
specificity
of
chromatographic
compounds
with
monstrated measured
the method
steps;
two of
the epimeric
compounds
with
authentic
peak
sample
to GLC
was
(Figl).The
subjected separation
in table
2 where
spect
to testosterone
Table
2: Relative
relative
time
of
when
time
1.0
1.0
1.0
1.0
200r-OH-P
1.87
1.54
1.53
-
2Of3-OH-P
1.71
1.36
1.40
-
zau-W-P
1.25
-
-
-
was
de-
of the
demonstra-
the biological
with
the
standard
pregnanolones retention standard
is demon-
times are
(Rt) during
testosterone
Testosteron,
was
seve-
tracer
identity
material
together
as internal
retention
(Retention
The
increase
of epimeric
by
of added
pregnanolones
by radiogaschromatography.
ted by a symmetrical
strated
is determined
the identity
with
re-
compiled.
GLC
= 1)
1.0
1.0
1.0
1.84
1.70
1.56
-
-
1.59
1.42
-
-
-
-
-
957
.55
.5?
1.22
1.15
1.0
1.0
%-W-P
.61
957
.59
029
.58
.52
3n-5o-P
.59
.5?
.59
.30
.59
.55
.5?
.54
.5?
3lz-513-P
-68
.58
.61
-66
.5?
-57
.85
.68
.?4
.81
.?2
.58 l?2
.61
3a-5g-P
.52 .34
.go
.?l
3g-AK43
.84
‘68
.?l
.48
.?8
.65
.66
.80
l66
71
This ment
1 by measuring
rivativea ment
criterion
one
total
uteru8
columns (960
purified
of the indicated
retention strated
time under
completed
GLC.
g wet weight)
as the authentic
different In this
was
are
with
standarda
conditions
de-
experi-
extracted
same
could
and
In
compiled,
the
The
relative
be demon-
of GLC.
n&g
Compound
Table
in experi-
chromatography.
experiment
compounde
six different
using
during
by additional
2 the rerrulte of this
presence
and
the 8p111efractions
and different
the extracts table
W~IB checked
3R-5u-P
+
+
+
+
+
4
18.7
h-&-P-S04
+
+
+
+
+
+
35.6
3&5&P
_
-
-
-
_
-
-
3S_5S_P_SO4
_
-
-
-
-
_
.
32..ggl-P
+
+
+
+
+
+
38.7
3a-5cr-P-SO4
+
+
+
+
+
+
9.4
3a-5a_p
_
_
_
_
*
-
-
3nL5%P-SO4
*
+
+
+
+
+
15.2
3l3-A5-P
+
+
+
+
+
+
25.8
313-A5-P-SO4
+
+
+
+
+
+
a.9
3:
Exo.1: Isolation of Pregnanolones from myometrium (970 g + indicate8 demonstration tiabue, 40. week of pregnancy). of compound with identical relative retention time ae the reference compound (these values are shown in table 2) a symmetrical peak increase after injection of authentic compound8 together with analysis sample.
S
72
Concentration
of metabolites
In experiment could
from
(k-%-P)
(3&5c+P), fate,
and
and
addition
3)
the
three
tissue.
tissue:
epimeric
as
the
free
compound
3a-sulfoxy-50-pregnan-20-one there
its
sulfate.
not
be
was
The
found.
compounds
a-hydroxy-W-pregnan-
3B-hydroxy-w-pregnan-20-one
present
both
in uterine
1 (table
be isolated
20-one
TREOXDI
and
(50-P-3&-S04).
3D-hydroxy-5-pregnan-20-one
epimer
30-5o-P
with
was
as sul-
metabolite
and could
3Lb5l3
the configuration
the main
In
(d5-P)
in this
uterus. In experiments ted. the
small
samples
each
same
uterus.
In table
oompiled. 35-k-P
The and
centration only was
2-21
Two
from not
In a water
blank
detected
titative (Exp.
of are
compounds
samples
were
the
processed
by
which
in
which
In the other
determination
extrac-
parts
results
or below
peaks
were
different
4 the quantitative
sulfoconjugate
detected
samples
from
to 296 rig/g tissue.
6 samples.
were
tissue
taken
predominant
its up
were
these
were
present
3a-5a-P
was
specimens
sensitivity
this of
the described
could
interfere
of the different
in con-
isolated steroid
the method. method
with
no
the quan-
pregnanolones
22). Progesterone
determined
also.
concentrations the reported The
could between
The
range
results
one
tissue
included were
the nonpregnant
amount of
be detected,
samples
in table
found
in the
uterus
of progesterone
the saturated There
the concentration
metabolites
are
steroids
these
were
4. The
to be within
(5).
from
a small nor
~OCY-OH-P in
of these
tissue
contained 2OeOH-P
and
was
no
but
2 and
3)
neither
progesterone statistical
of progesterone
tissue.
(Exp.
metabolites
correlation and
those
of
the
poetov. Ph. postov.Ph.
IO. 10.
Il.
2 3
4 5
6 7
18. 18.
18. 18.
18. 18.
24. 24.
41. 41.
12
13
14 15
‘16 17
18 19
20 21
22
Water
12. 12.
11
IO
5ml
5::
;::
;::
;:::
5.4 5.2 I
;::
-
28.1 17.5
23.8 36.0
114.4 75.9
121.1 116.9
106.7 81.2
%*43.
135.6 90:2
;::
11.
11.
x
zx
z
40.4 18.9
5.2 5.3
55.8 11.5
11.7
6.9 12.2
20.7 23.9
Z:;
37.2 20.4
7.8 11.7
2:: 245
I
52.7 40.6 50.6 81.8
10.0 10.5
120.9 33.1
10.0
35.2 10.0
10.0 110.8
72.5 30.3
9.4
22.6
18.6 12.4
18.7
128.5 110.6
I
5ty-P-3f3-so4
Wet weight)
10.0
32.4 30.4
32.3 38.0
21.2 61.4
23.4 52.9
35.0 41.9
249.1 296.4
10.0
22.5
24.6 19.2
38.7
(na;/g
3u-5a-P
muSCle
3i3-5qs-P
uterine
20 Cy-OH-P
in
I de teranined ;:;
not
4.9 5.2
970.0
Prog.
and metabolites
11.
blank
40.
1
Tissue (g)
4% Progesterone L
Pxp. Week of Pregnancy lo.
Pablo
24.0 13.3
12,9
IO
35.6
5~P-srrso4
DISCUSSION Assuming organ
that metabolism
is related
to hormone
of a hormone action
gained
by determining
the concentration
parent
steroid
tissue
From
in the
the 20 isomeric
from
progesterone
four
which
special
are
reduced
shown
to occur
ring
A was
found
target
By
tissues
these
be formed
depending
The
quantitative
ses
fundamentally
3 and
any
which
tissue may
steps
on
four
the enzyme
determination the
separation
the
molecule
5 only
the
are
which
saturation
be regarded
of has of
as so
(14).
epimeric
compounds
patternmin
of
of
be
can originate
steroid
of progesterone
metabolic
only
to 20q-reduction
in almost
target
could
the metabolites.
which
at positions
in organs
not of
of the
In contrast
interest.
also
compounds
by reduction
been
called
but
in its
information
these
may
the cells.
metabolites
of epimers
and
suppo-
sensitive
detection. Both and
criteria
electron
Adlercreutz the
tion
by GLC.
of TMSX
these
and
and
liquid
These
to about
chromatography
Chamberlain
& Thomas
SjZSvall (17) have
the quantitative
derivatives
methods
by gas
detection. (16)
et al.
separation
nanolones
are met
capture
authors
100 ng
reported
determination
however
which
limits steroid
used the
(15), on
of preg-
flame
ionisa-
sensitivity
in biological
of mate-
rial. Concerning nes
and
tion Molen
electron
separation
fs available. & Groen
progesterone
nanolones. these
(8) for
As
it appears
detection by GLC
the quantitative
electron
by
that
capture
the relative
as compared the
of pregnanololittle
to the method
smaller
of van
derivative detection
retention
to TMSI
informa-
determination
the monochloroacetyl
and
shown
derivatives
columns
In analogy
we used
gaschromatography
capture
of derivatives
ethers
derivatizing
of for
of preg-
times on
der
of
the same group
of
the monochloroacetate epimers sated and
during
however
longer
tection
les
to buffer
of uterine
GLC-methods This thod
by
are within
the
which
conditions
been
with
samples
of step
biological range
specificity
samp-
of other
of the me-
could
with
interfere
results
elithe
of experiment
of the method
could
with
3 derivatives
1
as the
be determined
of the method
demonstrated of human
it can be
metabolites
in the
under
on 3 different
that
were
In similar
the tissue. was
The
myometrium
found
Though phase
The
in uterine
isolated
range
concentration
comparable
determination.
and
From
these
during
preg-
3a-hydroxy-5aand in the was
iso-
as progesterone
in
of 3a-hydroxy-5a-pregnan-
to 20a-hydroxy-k-pregnen-3-one. if present
by TLC epimer
the samples
before
with
tissue
from human
of cycle
and
a8 "free** steroids
quantitative
eliminated
2-21).
3I3-hydroxy-5a-pregnan-20-one
3a-hydroxy-5D-pregnan-20-one as it was
experiment
(Exp.
in human
found
fraction.
mate-
!ja-reduced progesterone
30-hydroxy-Sa-pregnan-20-one
"sulfaten
to biological
same
myometrium
stated,
are present
pregnan-20-one
tory
amount
the
phases.
has
been
of
steps
The
pregnanolones
rial
not
of de-
chromatographic
specificity
applicability
20-one
with
similar
the
the different
The
lated
for
determination.
six differnt stationary
also
compounds
epimeric
nancy.
of known
as demonstrated
true
support
results
sensitivity
(QF-1)
(17). holds
quantitative
three
phase
and precision
by addition
and
other
further
the
lr5.
muscle
provided
minating
hand
of
can be compen-
stationary
reproducibility
accuracy,
effect
a selective
On the other
columns.
as determined
raids
the separation This
chromatography. by using
ie increased
The method
diminishes
was
not
the gaschromatographic
the configuration
and
determined
to our knowledge
3ib513 has
was
never
tissue. from
contained
a uterus none
of
of the postovulathe saturated
meta-
S
76 bolites
a correlation
progesterone
or
lones
tissue
in
the
The rone could
be
between
could
from
trapped
during
side
in
tively rine
higher muscle
these
of
blood
as
and
their be
metabolites in
of
in (17)
Sjiivall
contribution
progeste-
this
tissue
cannot
on
determined
by
pregnano-
of
blood
concentrations
reported
cannot
and
originate
as
endogenous
established.
circulation The
maternal and
be
myometrium
definitively.
experiments
not
whether
answered and
concentrations
20q-hydroxy-4-pregnen-3-one
question
isolated
TmEIEOXDI
to
the
the
or
be
fetal
preliminary are
compara-
amount
in
ute-
excluded.
ACKNOWLEDGEMENTS P.
I thank Dr. Munich University, The skilful graphy. gelt is gratefully
Unterburger, I. Dept. Int. Med. of who performed the radiogaschromatotechnical asistance of Miss P. Fiinfacknowledged.
The study was supported Forschungsgemeinschaft,
by Mi
the Deutsche 109/4
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