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Levels of 13,14-dihydro-15-keto-PGF2α in biological fluids as measured by radioimmunoassay
LEVELS
OF 1&14-DIHYDRO-l5-KETO-PGF2o AS MEASURED
Lawrence
Levine+
IN BIOLOGICAL
FLUIDS
BY RADIOIMMUNOASSAY*
and Rose Mary Gutierrez-Cernosek'
From the Graduate Brandeis University,
Department of Biochemistry Waltham, Massachusetts 02154
ABSTRACT Antibodies were prepared olite
directed
toward
in rabbits.
is immunodominant.
to a lesser extent, hydroxyl
lj,l&dihydro-15-keto-PGF20
The C-15 keto group The antibodies
the reduced
group and the C-11 hydroxyl
previously
C-15
keto group and the lj,l&double
netabolite
were
of biological cation
described
equally
samples
antibodies
of either
have been
Accepted
April
the
bond of the homologous Therefore, permits
metabolite.
metabolites
assay
identifi-
The
in human peripheral
sera and the levels of cross reacting
although the C-9
to 15-keto-PGF20
with both antisera
and quantitation
bond,
group of the metabolite.
immunodominant.
of lj,14-dihydro-15-lteto-PGF2c
recognize,
lj,l&double
With
of the metab-
levels venous in urine
determined.
25
*
This work was supported in part by a grant rrom the AmerIt is publication ican Cancer Society (Grant No. lC-LOL). JO. 852 from the Graduate De_uartment or Biochemistry, Brandeis University. .I. American Cancer Society Professor of Biochemistry (Award X0 . XP-21).
.$
Supported by Training Grant No. CA-05174 Cancer Institute, N. I. H.
JUNE
1973
VOL. 3 NO. 6
from the National
785
PROSTAGLANDINS
Radioimmunoassays for the determination of prostaglandins (PGs) of the E (l), A (2,3) and F (h-10) types have been developed.
Antibodies directed toward the two initial metabolites
of PGF2,, 15-keto-PGF2c (11) and 13,14-dihydro-15-keto-PGF2c (12), have also recently been prepared.
The latter metab-
olite is present in plasma at a concentration 20-30fold higher than the administered PGF2,, as measured by mass spectroscopy (12,13).
Except
for the report (12) of a single serum
analysis obtained by radioimmunoassay
(O-O.24 ng/ml),
levels of the 13,14-dihydro-15-keto-PGF2c
metabolite
endogenous in periph-
eral venous sera are unknown. We here report the serologic specificity of antibodies prepared in rabbits by immunization with polylysine-13,1&dihydro15-keto-PGF2c conjugates complexed to succinylated hemocyanin and their use in determining the levels of lj,l&dihydro-15keto-PGF20 in peripheral sera and in estimating PGF production in man. MATLRIALS
AND METHODS
The 13,14-dihydro-15-keto-PGF2u J. .&. Pike and U. Axen Michigan. following
The metabolite
and adjusted
methylaminopropyl) was allowed
was coupled
which
carbodiimide*HCl
in whose
synthesized.
0.005 M phosphate
NaCl.
786
were complexed
with
these antiqenic dialysis
against
of succinylated
to 2 ml with 0.14 M
2 ml of complete
received
con-
pH 7.0, 2.0 mg of the
an equal amount
were added
Each of two rabbits
We thank
overnight.
exhaustive
buffer,
and the volume was adjusted
To this mixture
adjuvant.
were added and the mixture
laboratory
After
jugates were
8
in 0.4 ml of
Eight mg of 1-ethyl-3-(3-di-
to stand at room temperature
0.15 !i NaC1, conjugate
by the
and mixed with
had been dissolved
to pH 7.2.
Dr. Helen Van Vunakis
hemocyanin
from Drs.
Kalamazoo,
to poly(L-lysine)
in 0.4 ml of dimethylformamide
mg of polylysine*HBr, water
obtained
Company,
Eight mg of 13,14-dihydro-15-keto-PGF2o
procedure.
were dissolved
was
of the Upjohn
Freund's
1 ml of this mixture
JUNE
1973
VOL.
3 NO. 6
PROSTAGLANDINS
via toe pad and intramuscular tion procedure
with
later.
The rabbits
jection
and each succeeding
week
a booster
injection,
[ 3HlPGF2a was prepared fraction
were used
were again
Boston,
for binding
with
13,14-Dihydro-15-keto-
by incubation Mass.)
of a dog lung homogenate
of [3~]~~~20, (New ting-
with
the cytoplasmic
(14).
has been reported
15-keto-[3H]PGF2a
the same as for
and the rabbits
PG metabolites
Corp.,
in-
Two weeks
week.
anti-13,14-dihydro-l5-keto-PGF2c.
land Nuclear
one week
for seven weeks.
was given
injection,
bled each succeeding Two tritiated
The same immuniza-
were bled one week after the second
after the last bleeding the primary
injection.
the same dose was repeated
The preparation
earlier
(11).
of
The ex-
tent of inhibition i3H]PGP2c
of the binding of 13,14-dihydro-15-ketoand 15-keto-[ 3H]PGF2c with anti-lj,l4-dihydro-
15-keto-PGF2c
by various
of inhibition
was the same when the
both metabolites
was
[ 3HlPGF2a prepared described
PGs was similar
Therefore,
identical.
chemically
and the sensitivity
['H]PGF2o
was used
used to prepare
the l+keto-
for most of the studies
in this report.
Radioimmunoassays
were performed
Rabbit
as follows.
antiserum diluted 1:50 in 0.01 M Tris, 0.14 M NaCl, 5.0 X -4 -4 M CaC12, pH 7.5, containing O.l,,, M MgS04, 1.5 X LO 10 gelatin,
was incubated
with
12,000 cpm) in the presence
the mixture
antibody-bound
precipitate
Bray's
PGs for
,nixture was
of goat anti-rabbit-
overnight
was collected
at approximately
was dissolved
in a modified
addition
was incubated
15-keto-L3H]PG
by centrifugation
(approximately
of unlabeled
of the reaction
After
0.3 ml or 0.4 ml.
y-globulin,
absence
The total volume
1 hr at 37O. either
L5-keto-[3H]PG or
at 2-4O.
The
as a precipitate
1,000 X g for j0 min.
The
in 0.2 ml of 0.1 N NaOH and counted
solution
in a Packard
liquid
scintillation
spectrometer.
JUNE 1973
VOL. 3 NO. 6
787
PROSTAGLANDINS
RESULTS Of the two rabbits keto-PGF2c binding
antigenic
antibodies
immunized
conjugate,
two weeks
tion and has continued
bleedings
studies
reported
precipitated
here were performed
section.
but only
This
[3~]~~~2a
homogenates
conditions,
rabbit
antiserum
that had been
or treated
benzoguinone
serum, as measured
with
reacts
The saturated
hapten,
is clearly
The
Alkaline
treatment
leads to dehydration inhibition
PGF2c3
788
to some extent group,
lOOfold;
ineffectThe C-9
since, despite
inhibition
by 15-keto-
less effective
than by 15-ketowhich probably
and loss of the C-11 hydroxyl whereas
alkaline
being
group,
treatment
its cross reactivity.
on the 13,1&-dihydro-15-keto-PGF2c
to the specificity
the most dominant
cross
role of the
of 15-keto-PGZ2,
does not affect
that four subsites
is not strikingly metabolite
from the relatively
of the 15-keto
PGE 2 is three to four times
15-keto-PGF2c
bond
(about 2$ cross reaction).
can be recognized
the immunodominance
is shown in
binding),
immunodominant
evident
by PGF2c
group
anti-
13,14-dihydro-15-keto-PGF2c,
13,1&double
ive inhibition
contribute
tissue
binding
of 15-keto-[3H]PGF2c
since the 15-keto-PGF2c
hydroxyl
creases
[3H]PGF2c
with certain
with the anti-13-l&dihydro-15-keto-
fairly extensively.
15-keto-group
PGF2,.
in the
did not bind
of the 1x,14-dihydro-15-keto-PGF2c
most effectively
immunodominant, reacts
that
2,3-dichloro-5,6-dicyano-1,4-
by inhibition
The homologous
PGF2,.
as described
incubated
(or 13,14-dihydro-15-keto-[3H]PGF2c 1.
All of the
a bleeding
(DDQ) (11).
The specificity
Fig.
hapten.
with
33%
bleeding,
4,000 cpm out of 12,000 cpm of 15-keto-[3H]PGF2c
under our radioimmunoassay Methods
In
2 kl precipitated
in a subsequent
metabolite;
injec-
The sec-
to the metabolite.
#737,
702 of the radiolabeled
2 ~1 precipitated
produced
primary
such antibodies.
antibodies
of rabbit
of the added tritiated
(8737)
one rabbit
after the initial
to produce
ond has not yet produced the earlier
with the 13,14-dihydro-15-
de-
of
It appears molecule
of anti-1x,14-dihydro-15-ketothe C-15 keto group.
JUNE 1973
Less dom-
VOL. 3 MD. 6
PROSTAGLANDINS
Rabbit
Anti-l3,14-Dihydro-15-Keto-PGF2,
13,14-Dihydro-l5-Keto-PGF
.j/ II) .x = ile
60
40
I5-Keio-PGEg
100
IO
I Nanograms
1000
Inhibitor
Fig. 1.
Inhibition of 15-keto-[%i]PGF20 rabbit anti-13,142 dihydro-15-keto-PGF2obinding. Each reaction mixture contained 0.1 ml of a I:50 dilution of rabbit antiserum, 0.1 ml of 15-
keto-[3H]PG (12,000 cpm), and 0.1 ml of unlabeled PG or PG metabolite dissolved in buffer (0.01 M Tris, 0.14 M NaCl, 5 x 10 -4 M MgSO,+, 1.5 x 10 -4 M caC12, O.l$ gelatin, pH 7.5) . Following incubation at 37O for 1 hr, 0.1 ml of goat anti-rabbit y-globulin was added and the mixtures were incubated overnight at 2-4O. The precipitate obtained from centrifugation (1000 x g for 30 min) was dissolved in 0.2 ml of 0.1 N NaOH and counted in a modified Bray's solution.
JUNE 1973
VOL. 3 NO. 6
789
inant but significant are the C-11 hydroxyl group, the C-9 hydroxyl group, and the saturated C-13,14 bond. Although the anti-13,14-dihydro-15_keto-PGF2c measures 13,14-dihydro-15-keto-PGF2o, it is not sufficiently specific to identify the inhibitor in an unknown sample.
For example, 50$ inhibition could reflect 4.0 ng of 13,14-dihydro-15-keto-
PGF2,, 7.0 ng of 15-keto-PGF2c, 35 ng of 15-keto-PGE2 or even 800 ng of PGF2,. The PGE metabolite can be eliminated from consideration by assaying the sample before and after alkaline treatment. PGF2o.
The PGF2c can be eliminated by assaying with anti-
(The latter antibodies do not cross react signifi-
cantly with the 15-keto-PGF2c metabolite.)
The contribution by 15-keto-PGF2c can be evaluated by assay with the antil+keto-PGF2o,
with which the 13,14-dihydro-15-keto-PGF2c
metabolites cross react about 5% (Fig. 2).
Therefore, with
the use of several antisera with various known specificities, =.q., anti-PGF2c, anti-15-keto-PGF2c, and anti-13,1&dihydro15-keto-PGF20, the composition of PGF and its primary metabolites can be identified and estimated. In sera and spinal fluids that we have already examined, the inhibition of the anti-13,14-dihydro-15-keto-PGF2c has been quantitatively accounted for by 13,14-dihydro-15-keto-PGF2c. Antibodies directed to both PGF2, and PGFla have previously been used to estimate levels of PGF2c in human peripheral venous sera.
In 78 samples assayed, males had more circulating PGF2,
in their blood than females, but there was a wide range in the values obtained from both males and females (males:
889 i
564 pg/ml serum: females:
For
272 z 221 pg/ml serum) (15).
these assays, the sera were treated with a methylal-ethanol system to denature the protein and release protein-bound PG into the organic phase.
After removal of the organic solvent
by evaporation, the material soluble in aqueous solvents was dialyzed against a small volume of an appropriately buffered aqueous solution and the dialysate was assayed for PGs. This procedure usually yielded 60-70~ recovery. Similar experiments
790
JUNE
1973
VOL.
3 NO. 6
PROSTAGLANDINS
Robbat
Antl-13.14-Dlhydro-l5-Keto-PGF2,
100 r
t 60
a i
Monkey
E ap
AntI-15-Keto-PGFp,
100
1 01
I
I
I
I
IO
100
Nanograms
Fig.
Inhibition
2 (top).
rabbit
are identical
(bottom). monkey
InhIbItor
of 15-keto-[3H]PGF20
anti-13,14-dihydro-l5-keto-PGF2o.
conditions
to those
Inhibition
anti-15-keto-PGF2o.
ml of a 1:50 dilution
I
1000
binding
described
in Fig.
of 15-keto-[3H]PGF20 Each reaction
of monkey
antiserum,
1.
binding
mixture
to
contained
PG or PG metab-
-4
in buffer (0.01 M Tris, 0.14 M NaCl, 5 X 10 -4 Following M CaC12, 0.176 gelatin, pH 7.5). 1.5 x 10
olite dissolved !“I
Mgs
0 4 ,
incubation globulin 2-4O.
0.1
0.1 ml of 15-keto-
(12,000 Cpm), and 0.1 ml of unlabeled
['H]PGF2d
to
The experimental
at j7O for 1 hr, 0.1 ml of rabbit
was added and the mixtures
The precipitate
obtained
for j0 min) was dissolved in a modified
JUNE 1973
Bray's
were
anti-monkey
incubated
from centrifugation
y-
overnight
at
(1000 X g
in 0.2 ml of 0.1 N NaOH and counted
solution.
VOL. 3 NO. 6
791
PROSTAGLANDINS
with 15-keto-PGF2c and 13,14-dihydro-15-keto-PGF2c also have led to 60-70s
recovery.
Using this method of extraction we
have assayed peripheral venous sera for PGF2,, 15-keto-PGF2c, and lj,l&dihydro-15-keto-PGF2c.
These levels are shown in
Fig. 3. The levels of the lj,l&dihydro-15-lceto-PGF2c metabolite are much greater than those of 15-keto-PGF2c or PGF2c in blood.
(The same serum samples were assayed with both anti-
metabolite sera (Fig. 2) to identify the metabolite being measured.) We have also measured the levels of 13,14-dihydro-15-ketocycle (Fig. 4). These values menstrual could not be correlated with ovulation (as measured by basal body temperature). Patients C and D were taking Norinyl (1+80 21-day)
PGF2, throughout the
and Gvral pills. Many of the metabolic products of PGF~~ found in urine have been identified (16,17).
Several of them (Fig. 5) have the
requisite structure (the four antigen subsites) for cross reaction with anti-13,14-dihydro-15-keto-PGF2c (the ga, lla-dihydroxy15-keto of the C-20 PG, the 7a, ga-dihydroxy-lj-keto of the C-18 and c-1.6 metabolites, and the 5a, 7a-dihydroxy-ll-keto of the C-14 metabolite).
Even some of the PGE metabolites could
cross react with this antiserum but to a lesser degree.
we
have analyzed several human urines, unextracted, for reaction The reactions
with antibodies to 13,14-dihydro-15-keto-PGF2a.
of 10 1_~1 of unextracted human urine with antisera to PGF2,, PGDl, 15-keto-PGF2a, and 13,14-dihydro-15-keto-PGF2a are shown in At the 10 ~1 level of urine, only the anti-13,14-
Table I.
dihydro-15-keto-PGF2c cross reacts significantly with metabolites present in urine. we can assign numbers for this serologic activity with anti-13,14-dihydro-15_keto-PGF2at if we assume that the c-14, c-16, and C-18 metabolites (Fig. 5) react as effectively as the C-20 metabolite. This estimate probably represents a minimal level of PGF production not Only on the basis of the above assumption but also because some PGF metabolites present do not have the requisite four subsites and therefore do not cross react.
792
JUNE 1973
VOL. 3 NO. 6
PROSTAGLANDINS
13,14-Dihydro-
I5-Kcto
I5-Kefo-PGF2,
-PGFza
PGF2.
.
IO
9
Fig. and
Levels
3.
seru-n,
represent
'-nean . The are
JUNE
of
in hulaan
DGE',,
also
1973
1~,14-dihydro-15-keto-PGF2a, sera.
the
mean
The
values,
and
the
lj,l4-dihydro-15-keto-PGF~~
15-lceto-PGFza,
expressed
standard _
as
ng/ml
deviation
levels
of
each
of
the
subject
;.ncluded.
VOL.
3 NO. 6
793
PROSTAGLANDINS
Fig. wonen
4.
pressed
794
Levels
throughout as ny/ml
of lj,14-dihydro-13-l;eto-PG1~LU the ,nenstrual cycle.
in sera of
The values
are ex-
serum.
JUNE 1973
VOL. 3 NO. 6
PROSTAGLANDINS
Fig.
5.
PGF2cz
metabolites
subjects
(16,17).
subsites
recognized
JUNE 1973
Enclosed
identified
in urine of female
areas contain
the four antigenic
by anti-13,14-dihydro-15_keto-PGF2a.
VOL. 3 NO. 6
795
PROSTAGLANDINS
The levels of PGF metabolite(s) males and 10 females
expressed
creatinine
in Table
duction
are given
of PGF/24
in the urines
of 11
both as 114124 hr and wg/g of II.
The minimal
hr would be around
value
for pro-
663-148 ILg for males and
j5-88 pg for females. We have also determined
the levels of metabolites
in rat
urines
that cross react with anti-lj,l4-dihydro-lj-keto-PGF
It was
found that in most rat urine
olites
that cross react with anti-lj,14-dihydro-15-keto-PGF and anti-PGBl
anti-15-keto-PGF2c,
All urines
(Table III).
reacted
samples
5 ~1 contained
but not with extensively
.
2a metab2aJ
anti-PGlr'2,(X
with
anti-lj,l4-
dihydro-15-keto-PGF2a
at the 10 1~1 level, but at tllis Level,
there was significant
cross reaction
with unextracted Table
rat urine)
IV are calculations
The daily production
from this inhibition as m
expressed
with respect
excretion,
The younger
PGF than man.
with
PGF metabolite/g
of
of PGF in the rat is much
less than in man, but when to creatinine
non-specific The data in
with anti-PGF2c.
obtained
5 ~1 of rat urine and are expressed creatinine.
(possibly
as a specific
activity
the rat produces
rats produce
more
more PGP than older
rats. DISCUSSION BecauSe
of the large number
quantitative measured bodies
and qualitative
groups
with
specificity
PGF compounds
is available.
toward
15-keto-PGF2o,
analysis
groups
antibodies
on
functional will
antibodies among PGFl,,
and 13,14-dihydro-
antibodies between
as
for some of the
distinguish
13,14-dihydro-15-keto-PGF20,
we have prepared
fluids,
by these procedures
of antibodies
easily
structures,
of anti-
functional
we have prepared
that do not distinguish
and PGF 2o (14).
on a battery
different
(1.5). We also have prepared
hydro-PGF20
796
PGF2, which
related
of biological
if these different
Such a library
be simplified.
PGF2,
toward
Of course,
are immunodominant,
directed
depend
by radioimmunoassay,
the PG molecules.
of closely
analyses
to lj,l4-di13,14-dihydro-PGF2a to PGFla
JUNE 1973
(PGFl,
VOL. 3 NO. 6
PROSTAGLANDINS
reacts
more effectively
than PGF2,).
bodies
to 15-keto-PGF2c
that react much more effectively
15-keto-PGF2c here,
than 13,14-dihydro-PGF2c
antibodies
tinguish
between With
PGF2,.
to 13,14-dihydro-l5-keto-PGF2c 15-keto-PGF2c
and identify
sample.
of different different
and PGF2,.
specificities,
compounds
with
As reported
do not dis-
we can begin
all of these PG compounds
Without
anti-
and 13,14-dihydro-15-keto-
the use of all of these antisera,
to guantitate biological
We have prepared
such a catalogue some physical
must be used prior
in a
of antibodies
separation
of the
to assay with a single
antiserum. As measured
by radioimmunoassay
with
in human sera have been measured. PGF2c metabolite, 13,14-dihydro-15-keto-PGF2,_, higher
than those of the metabolite,
This conclusion leagues
had been reached
(l2,l3), who quantitated
metabolite,
reported
analysis
by Samuelsson
than the administered
13, lb-wdro-15-keto-PGF2c than endogenous levels of PGF2,.
as measured
sera were reported average greater,
PGF2,.
4.8
ng/ml
(Fig. 3),
They
were
20-
Endogenous
be expected
levels of
to be higher
The only reported
endogenous
in serum is O-240 pg/ml although
less than 0.5
ng/ml
normal
in endogenous
in one
six other
(12).
Our
is considerably
and we even have observed
in a presumably
this discrepancy
and its
by mass
of PGF2,.
level of 13,14-dihydro-15-keto-PGF2c
as high as 26 ng/ml Despite
and his col-
in plasma
by radioimmunoassay, to have
and PGF2,.
the levels of PGF2,
also would
serum,
are significantly
after administration
for this major metabolite
and
15-keto-PGF2c,
that the levels of the latter metabolite
30fold higher
value
antisera,
The levels of the
13,14-dihydro-15-keto-PGF2c,
spectrometric
several
13,14-dihydro-15-keto-PGF2c
levels of 15-keto-PGF2c,
serum
levels
a value
(Fig. 4~).
in blood
of
13,14-dihydro-15-keto-PGF2c,
the levels of 13,14-dihydro-15-keto-
PGF2,
much better
reflect
of PGF2,
PGF production
or the initial
surprising,
JUNE 1973
metabolite,
since many tissues
VOL. 3 NO. 6
than do the blood
15-keto-PGF2o.
have high
15-hydroxy
This
levels
is not
PG dehydro-
797
PROSTAGLANDINS
genase
and 15-keto-PG-A13,14
With
reductase
such high concentrations
activities
(18).
ofQ,l&dihydro-15-keto-
in sera, could the PGF2, levels previously obtained PGF2a radioimmunoassay merely reflect cross reaction of 13,14with anti-PGF20?
dihydro-15-keto-PGF20
used in our laboratory, reacts
average
the anti-PGF20
13,14-dihydro-15-keto-PGF2c
Our PGF2,
less than l$.
er (6-l?“&) .
With
however,
suggests
level of 500 pg of 15-keto-PGF2o
13,14-dihydro-15-keto-PGF2o
cross
levels are considerably
The same reasoning,
average
(about g$ of the
level) reflects,
of the 13,14-dihydro-15-keto-PGF2o
anti-15-keto-PGF2o
(cross reaction
is 5$).
data are not sufficiently to calculate
of 15-keto-PGF2c
13,14-dihydro-15-keto-PGF2c
a constant
mass spectrometric production culated
have
(19).
istration
as measured
led to the assignment daily rates were
We have determined
of [3~]~~~2.
urinary
metabolites
PGF2o.
These
tion obtained
on analyses
with antiserum
calculations with
after admin-
a minimal
produc-
urine after demonstraurine did not inhibit
(Table I).
The daily produc-
from mass spectrometric
150-330
kg in males
data of the major urinary
metabolite,
were
and 20-40 bg in females.
our minimal
daily production
PGF, determined
66-148
spectrometry cedures
798
from the serologic
activities
of urine
kg in males and 35-88 pg in females.
tive analysis
of blood
of
made from the per cent inhibi-
tion rates of PGEl and PGE2, calculated
were
of
cal-
to 13,14-dihydro-15-keto-
of unextracted
non-specifically
by
of the cross reactions
10 ~1 of unextracted
tion that this quantity immune binding
were
ratio of
in each serum.
in urine,
These
our
at the low levels
from the yield of the major metabolites
tion rate for PGF based
with
or variable
to 15-keto-PGF2,
analysis,
rates to PGE
in part,
Unfortunately,
accurate
The levels of major metabolite
great-
that our
the cross reaction
analytical
by
samples,
As in compara-
levels of PG by radioimmunoassay
(20), the levels obtained
rates of
and mass
by the immunologic
pro-
are higher.
JUNE 1973
VOL. 3 NO. 6
PROSTAGLANDINS
REFERENCES 1.
Jaffe, B. M., J. W. Smith, W. T. Newton, and C. W. Parker. Radioimmunoassay for Prostaglandins. Science 171: 494, 1971.
2.
Stylos, W. A., and B. Rivets. Preparation of Specific Antiserum to Prostaglandin A. Prostaglandins 2: 103, 1972.
3.
Zusman, R. M., B. V. Caldwell, L. Speroff, and H. R. Behrman. Radioimmunoassay of the A Prostaglandins. Prostaglandins 2: 41, 1972.
4.
Levine, L., and H. Van Vunakis. Antigenic Activity of Prostaglandins. Biochem. Biophys. Res. Commun. 41: 1171, 1970.
5.
Levine, L., R. M. Gutierrez-Cernosek, and H. Van Vunakis. Specificities of Prostaglandins Bl, F AntigenAntibody Reactions. J. Biol. Chem. 24@t:a$8F2$5971.
,0.
Caldwell, B. V., S. Burstein, W. A. Brock, and L. Speroff. Radioimmunoassay of the F Prostaglandins. J. Clin. Endocr. 33: 171, 1971.
7.
Kirton, K. T., J. C. Cornette, and K. L. Barr. Characterization of Antibody to Prostaglandin F20. Biochem. Biophys. Res. Commun. 47: 903, 1972.
8.
Orczyk, G. P., and H. B. Behrman. Ovulation Blockade by Aspirin or Indomethacin. In vivo evidence for a Role of Prostaglandin in Gonadotrosii??&retions. Prostaglandins 1: 3, 1972.
9.
Gutierrez Cernosek, R. M., L. M. Morrill, and L. Levine. Levels in Peripheral Sera of Man. Prostaglandin F Prostaglandins S? 71, 1972.
10. Yu, s. c., and G. Burke. Antigenic Activity of Prostaglandins: Specificities of Prostaglandins E , A and F2o AntigenAntibody Reactions. Prostaglandins 2: $1, 1972. 11. Levine, L., and R. M. Gutierrez-Cernosek. Preparation and Specificity of Antibodies to 15-keto-prostaglandin F20. Prostaglandins 2: 281, 1972. .. 12. Granstrom, E., and B. Samuelsson. Development and Mass Spectrometric Evaluation of a Radioimmunoassay for 9a, lladihydroxy-15-ketoprost-5-enoic Acid. FEBS Letters 26: 211, 1972. 13. Samuelsson, B. Endogenous Synthesis of Prostaglandins. Third Conference on Prostaglandins in Fertility Control, WHO Research Institutet, Stockholm, 1972, p. 189.
JUNE 1973 VOL. 3 NO. 6
799
PROSTAGLANDINS
14.
Levine, L. Antibodies to Pharmacologically Active Molecules: Specificities and Some Applications of AntiProstaglandins. Pharmacological Reviews, in press.
15.
Gutierrez-Cernosek, R. M., and L. Levine. Serological Aspects of Prostaglandins. J. Reproductive Med., in press.
16.
Granstrgm, E., and B. Samuelsson. On the Metabolism of in Female Subjects. II. Structures of ;yOs;~~;;~;;:,F,ea . J. Biol. Chem. 246: 7470, 1971. ,I Granstrom, E. on the Metabolism of Prostaglandin F in E'emaleSubjects. Structures of Two Cl4 Metabolites?a sur. J. Biochem. 25: 581, 1972.
17.
18.
Samuelsson, B., E. Granstrgm, K. Green, and M. Hamberg. Metabolism of Prostaglandins. Ann. N. Y. Acad. Sci. 180: 138, 1971.
19.
Hamberg, M. Inhibition of Prostaglandin Synthesis in Man. Biochem. Biophys. Res. Commun. 49: 720, 1972.
20.
Sanuelsson, B., Convener. Round Table Discussion. vances in Biosciences 9: 1973, in press.
800
JUNE 1973
Ad-
VOL. 3 NO. 6
PROSTAGLANDINS
Table I. Inhibition of Prostaglandin Antiprostaglandin Reaction by 10 ~1 Human Urine
sub-
ject 1 2
3 4 5 6 7 8
9 10 11 12
l3 14 l5 16 17 18
19
Anti-13,14dihydrol+ketoPGF2, (S) 21
l9 15 29 17 25 9 20 18 11
17 l9 l5 l5 l9 24
29 13 10
JUNE 1973 VOL. 3 NO. 6
Anti-l+ keto-PGF2a ($1
6 6 0 5 0 0
AntiPGF2, -@J-m 1
7 3 3 0 0
1
AntiPGBl 7
5 3 1 7 3 0
0 0 0 0 0
0 0 0
3
1
0 0 0 0
5 6 0
2
2
0
5
3
2 2
3 7 1 0 4
4
0
0
3
0
4
0
3
801
PROSTAGLANDINS
Table If.
Levels
of 13,1&Dihydro-15-Keto-Prostaglandin
Metabolites*
in Human Urine Females
Males Subject
yg/24 hr
cLg/g creatinine
Subject
kg/24 hr
clg/g creatinine
1
147
85
1
88
2
107
52
2
80
58 68
3
121
55
4
66
38
3 4
85 45
58 41
5
80
65
5
35
26
6
82
45
6
59
43
7
78 89
35 41
7 8
40 50
37 52
9 10
70 52
50 5a
-
-
8
9
86
44
10
126
64
11
148
35
49 I 12 60 " 19 103 + 29 52 I 15 Mean + S.D. *., Metabolic products that cross react with anti-lj,l&dihydrol+keto-PGF2a.
802
JUNE 1973
VOL. 3 NO. 6
PRO.STAGLANDIN.9
Table
III.
Inhibition
Prostaglandin
Anti-13,14dihydro-15keto-PGF2a
Rat
+* 8
of Prostaglandin-Anti-
Reactions
by
5 ~1 of Rat Urine
Anti-l+ keto-PGF2c
AntiPGF2cl 03 0
($1
($1
35
13
40
17
36
15
5 0
34 33
13 12
0
35
12
35
15
30
1
AntiPGBl -7K 4 5 N.D.* 5 1 5
3 6
17
3
7
9
23
1-5 11
5
5
10
28
12
0
5
11
31 16
14
5 2
11
0
13 14
16
7 6
31
11
2
3 10
15 16
30
13
2
12
17
7
5
4
12
2
*
** 1 ~1 of this rat N.D. = not determined. urine was used in the assay reaction mixture.
JUNE
1973
VOL.
3 NO. 6
803
PROSTAGLANDINS
Table
Iv.
Levels of 13,14-Dihydro-15-Keto-
Prostaglandin
Metabolites
in Urine
of Female Rats Rat
1-LgPG/g creatinine 354
122
241 520
204 224
421
250
347
139
425
271
342
203
284
804
kg PG/g creatinine
(250
7xEq
Mean +, S.D.
Rat
367 +_ 88
125 192 “_ 57
JUNE 1973
VOL. 3 NO. 6
OF 1&14-DIHYDRO-l5-KETO-PGF2o AS MEASURED
Lawrence
Levine+
IN BIOLOGICAL
FLUIDS
BY RADIOIMMUNOASSAY*
and Rose Mary Gutierrez-Cernosek'
From the Graduate Brandeis University,
Department of Biochemistry Waltham, Massachusetts 02154
ABSTRACT Antibodies were prepared olite
directed
toward
in rabbits.
is immunodominant.
to a lesser extent, hydroxyl
lj,l&dihydro-15-keto-PGF20
The C-15 keto group The antibodies
the reduced
group and the C-11 hydroxyl
previously
C-15
keto group and the lj,l&double
netabolite
were
of biological cation
described
equally
samples
antibodies
of either
have been
Accepted
April
the
bond of the homologous Therefore, permits
metabolite.
metabolites
assay
identifi-
The
in human peripheral
sera and the levels of cross reacting
although the C-9
to 15-keto-PGF20
with both antisera
and quantitation
bond,
group of the metabolite.
immunodominant.
of lj,14-dihydro-15-lteto-PGF2c
recognize,
lj,l&double
With
of the metab-
levels venous in urine
determined.
25
*
This work was supported in part by a grant rrom the AmerIt is publication ican Cancer Society (Grant No. lC-LOL). JO. 852 from the Graduate De_uartment or Biochemistry, Brandeis University. .I. American Cancer Society Professor of Biochemistry (Award X0 . XP-21).
.$
Supported by Training Grant No. CA-05174 Cancer Institute, N. I. H.
JUNE
1973
VOL. 3 NO. 6
from the National
785
PROSTAGLANDINS
Radioimmunoassays for the determination of prostaglandins (PGs) of the E (l), A (2,3) and F (h-10) types have been developed.
Antibodies directed toward the two initial metabolites
of PGF2,, 15-keto-PGF2c (11) and 13,14-dihydro-15-keto-PGF2c (12), have also recently been prepared.
The latter metab-
olite is present in plasma at a concentration 20-30fold higher than the administered PGF2,, as measured by mass spectroscopy (12,13).
Except
for the report (12) of a single serum
analysis obtained by radioimmunoassay
(O-O.24 ng/ml),
levels of the 13,14-dihydro-15-keto-PGF2c
metabolite
endogenous in periph-
eral venous sera are unknown. We here report the serologic specificity of antibodies prepared in rabbits by immunization with polylysine-13,1&dihydro15-keto-PGF2c conjugates complexed to succinylated hemocyanin and their use in determining the levels of lj,l&dihydro-15keto-PGF20 in peripheral sera and in estimating PGF production in man. MATLRIALS
AND METHODS
The 13,14-dihydro-15-keto-PGF2u J. .&. Pike and U. Axen Michigan. following
The metabolite
and adjusted
methylaminopropyl) was allowed
was coupled
which
carbodiimide*HCl
in whose
synthesized.
0.005 M phosphate
NaCl.
786
were complexed
with
these antiqenic dialysis
against
of succinylated
to 2 ml with 0.14 M
2 ml of complete
received
con-
pH 7.0, 2.0 mg of the
an equal amount
were added
Each of two rabbits
We thank
overnight.
exhaustive
buffer,
and the volume was adjusted
To this mixture
adjuvant.
were added and the mixture
laboratory
After
jugates were
8
in 0.4 ml of
Eight mg of 1-ethyl-3-(3-di-
to stand at room temperature
0.15 !i NaC1, conjugate
by the
and mixed with
had been dissolved
to pH 7.2.
Dr. Helen Van Vunakis
hemocyanin
from Drs.
Kalamazoo,
to poly(L-lysine)
in 0.4 ml of dimethylformamide
mg of polylysine*HBr, water
obtained
Company,
Eight mg of 13,14-dihydro-15-keto-PGF2o
procedure.
were dissolved
was
of the Upjohn
Freund's
1 ml of this mixture
JUNE
1973
VOL.
3 NO. 6
PROSTAGLANDINS
via toe pad and intramuscular tion procedure
with
later.
The rabbits
jection
and each succeeding
week
a booster
injection,
[ 3HlPGF2a was prepared fraction
were used
were again
Boston,
for binding
with
13,14-Dihydro-15-keto-
by incubation Mass.)
of a dog lung homogenate
of [3~]~~~20, (New ting-
with
the cytoplasmic
(14).
has been reported
15-keto-[3H]PGF2a
the same as for
and the rabbits
PG metabolites
Corp.,
in-
Two weeks
week.
anti-13,14-dihydro-l5-keto-PGF2c.
land Nuclear
one week
for seven weeks.
was given
injection,
bled each succeeding Two tritiated
The same immuniza-
were bled one week after the second
after the last bleeding the primary
injection.
the same dose was repeated
The preparation
earlier
(11).
of
The ex-
tent of inhibition i3H]PGP2c
of the binding of 13,14-dihydro-15-ketoand 15-keto-[ 3H]PGF2c with anti-lj,l4-dihydro-
15-keto-PGF2c
by various
of inhibition
was the same when the
both metabolites
was
[ 3HlPGF2a prepared described
PGs was similar
Therefore,
identical.
chemically
and the sensitivity
['H]PGF2o
was used
used to prepare
the l+keto-
for most of the studies
in this report.
Radioimmunoassays
were performed
Rabbit
as follows.
antiserum diluted 1:50 in 0.01 M Tris, 0.14 M NaCl, 5.0 X -4 -4 M CaC12, pH 7.5, containing O.l,,, M MgS04, 1.5 X LO 10 gelatin,
was incubated
with
12,000 cpm) in the presence
the mixture
antibody-bound
precipitate
Bray's
PGs for
,nixture was
of goat anti-rabbit-
overnight
was collected
at approximately
was dissolved
in a modified
addition
was incubated
15-keto-L3H]PG
by centrifugation
(approximately
of unlabeled
of the reaction
After
0.3 ml or 0.4 ml.
y-globulin,
absence
The total volume
1 hr at 37O. either
L5-keto-[3H]PG or
at 2-4O.
The
as a precipitate
1,000 X g for j0 min.
The
in 0.2 ml of 0.1 N NaOH and counted
solution
in a Packard
liquid
scintillation
spectrometer.
JUNE 1973
VOL. 3 NO. 6
787
PROSTAGLANDINS
RESULTS Of the two rabbits keto-PGF2c binding
antigenic
antibodies
immunized
conjugate,
two weeks
tion and has continued
bleedings
studies
reported
precipitated
here were performed
section.
but only
This
[3~]~~~2a
homogenates
conditions,
rabbit
antiserum
that had been
or treated
benzoguinone
serum, as measured
with
reacts
The saturated
hapten,
is clearly
The
Alkaline
treatment
leads to dehydration inhibition
PGF2c3
788
to some extent group,
lOOfold;
ineffectThe C-9
since, despite
inhibition
by 15-keto-
less effective
than by 15-ketowhich probably
and loss of the C-11 hydroxyl whereas
alkaline
being
group,
treatment
its cross reactivity.
on the 13,1&-dihydro-15-keto-PGF2c
to the specificity
the most dominant
cross
role of the
of 15-keto-PGZ2,
does not affect
that four subsites
is not strikingly metabolite
from the relatively
of the 15-keto
PGE 2 is three to four times
15-keto-PGF2c
bond
(about 2$ cross reaction).
can be recognized
the immunodominance
is shown in
binding),
immunodominant
evident
by PGF2c
group
anti-
13,14-dihydro-15-keto-PGF2c,
13,1&double
ive inhibition
contribute
tissue
binding
of 15-keto-[3H]PGF2c
since the 15-keto-PGF2c
hydroxyl
creases
[3H]PGF2c
with certain
with the anti-13-l&dihydro-15-keto-
fairly extensively.
15-keto-group
PGF2,.
in the
did not bind
of the 1x,14-dihydro-15-keto-PGF2c
most effectively
immunodominant, reacts
that
2,3-dichloro-5,6-dicyano-1,4-
by inhibition
The homologous
PGF2,.
as described
incubated
(or 13,14-dihydro-15-keto-[3H]PGF2c 1.
All of the
a bleeding
(DDQ) (11).
The specificity
Fig.
hapten.
with
33%
bleeding,
4,000 cpm out of 12,000 cpm of 15-keto-[3H]PGF2c
under our radioimmunoassay Methods
In
2 kl precipitated
in a subsequent
metabolite;
injec-
The sec-
to the metabolite.
#737,
702 of the radiolabeled
2 ~1 precipitated
produced
primary
such antibodies.
antibodies
of rabbit
of the added tritiated
(8737)
one rabbit
after the initial
to produce
ond has not yet produced the earlier
with the 13,14-dihydro-15-
de-
of
It appears molecule
of anti-1x,14-dihydro-15-ketothe C-15 keto group.
JUNE 1973
Less dom-
VOL. 3 MD. 6
PROSTAGLANDINS
Rabbit
Anti-l3,14-Dihydro-15-Keto-PGF2,
13,14-Dihydro-l5-Keto-PGF
.j/ II) .x = ile
60
40
I5-Keio-PGEg
100
IO
I Nanograms
1000
Inhibitor
Fig. 1.
Inhibition of 15-keto-[%i]PGF20 rabbit anti-13,142 dihydro-15-keto-PGF2obinding. Each reaction mixture contained 0.1 ml of a I:50 dilution of rabbit antiserum, 0.1 ml of 15-
keto-[3H]PG (12,000 cpm), and 0.1 ml of unlabeled PG or PG metabolite dissolved in buffer (0.01 M Tris, 0.14 M NaCl, 5 x 10 -4 M MgSO,+, 1.5 x 10 -4 M caC12, O.l$ gelatin, pH 7.5) . Following incubation at 37O for 1 hr, 0.1 ml of goat anti-rabbit y-globulin was added and the mixtures were incubated overnight at 2-4O. The precipitate obtained from centrifugation (1000 x g for 30 min) was dissolved in 0.2 ml of 0.1 N NaOH and counted in a modified Bray's solution.
JUNE 1973
VOL. 3 NO. 6
789
inant but significant are the C-11 hydroxyl group, the C-9 hydroxyl group, and the saturated C-13,14 bond. Although the anti-13,14-dihydro-15_keto-PGF2c measures 13,14-dihydro-15-keto-PGF2o, it is not sufficiently specific to identify the inhibitor in an unknown sample.
For example, 50$ inhibition could reflect 4.0 ng of 13,14-dihydro-15-keto-
PGF2,, 7.0 ng of 15-keto-PGF2c, 35 ng of 15-keto-PGE2 or even 800 ng of PGF2,. The PGE metabolite can be eliminated from consideration by assaying the sample before and after alkaline treatment. PGF2o.
The PGF2c can be eliminated by assaying with anti-
(The latter antibodies do not cross react signifi-
cantly with the 15-keto-PGF2c metabolite.)
The contribution by 15-keto-PGF2c can be evaluated by assay with the antil+keto-PGF2o,
with which the 13,14-dihydro-15-keto-PGF2c
metabolites cross react about 5% (Fig. 2).
Therefore, with
the use of several antisera with various known specificities, =.q., anti-PGF2c, anti-15-keto-PGF2c, and anti-13,1&dihydro15-keto-PGF20, the composition of PGF and its primary metabolites can be identified and estimated. In sera and spinal fluids that we have already examined, the inhibition of the anti-13,14-dihydro-15-keto-PGF2c has been quantitatively accounted for by 13,14-dihydro-15-keto-PGF2c. Antibodies directed to both PGF2, and PGFla have previously been used to estimate levels of PGF2c in human peripheral venous sera.
In 78 samples assayed, males had more circulating PGF2,
in their blood than females, but there was a wide range in the values obtained from both males and females (males:
889 i
564 pg/ml serum: females:
For
272 z 221 pg/ml serum) (15).
these assays, the sera were treated with a methylal-ethanol system to denature the protein and release protein-bound PG into the organic phase.
After removal of the organic solvent
by evaporation, the material soluble in aqueous solvents was dialyzed against a small volume of an appropriately buffered aqueous solution and the dialysate was assayed for PGs. This procedure usually yielded 60-70~ recovery. Similar experiments
790
JUNE
1973
VOL.
3 NO. 6
PROSTAGLANDINS
Robbat
Antl-13.14-Dlhydro-l5-Keto-PGF2,
100 r
t 60
a i
Monkey
E ap
AntI-15-Keto-PGFp,
100
1 01
I
I
I
I
IO
100
Nanograms
Fig.
Inhibition
2 (top).
rabbit
are identical
(bottom). monkey
InhIbItor
of 15-keto-[3H]PGF20
anti-13,14-dihydro-l5-keto-PGF2o.
conditions
to those
Inhibition
anti-15-keto-PGF2o.
ml of a 1:50 dilution
I
1000
binding
described
in Fig.
of 15-keto-[3H]PGF20 Each reaction
of monkey
antiserum,
1.
binding
mixture
to
contained
PG or PG metab-
-4
in buffer (0.01 M Tris, 0.14 M NaCl, 5 X 10 -4 Following M CaC12, 0.176 gelatin, pH 7.5). 1.5 x 10
olite dissolved !“I
Mgs
0 4 ,
incubation globulin 2-4O.
0.1
0.1 ml of 15-keto-
(12,000 Cpm), and 0.1 ml of unlabeled
['H]PGF2d
to
The experimental
at j7O for 1 hr, 0.1 ml of rabbit
was added and the mixtures
The precipitate
obtained
for j0 min) was dissolved in a modified
JUNE 1973
Bray's
were
anti-monkey
incubated
from centrifugation
y-
overnight
at
(1000 X g
in 0.2 ml of 0.1 N NaOH and counted
solution.
VOL. 3 NO. 6
791
PROSTAGLANDINS
with 15-keto-PGF2c and 13,14-dihydro-15-keto-PGF2c also have led to 60-70s
recovery.
Using this method of extraction we
have assayed peripheral venous sera for PGF2,, 15-keto-PGF2c, and lj,l&dihydro-15-keto-PGF2c.
These levels are shown in
Fig. 3. The levels of the lj,l&dihydro-15-lceto-PGF2c metabolite are much greater than those of 15-keto-PGF2c or PGF2c in blood.
(The same serum samples were assayed with both anti-
metabolite sera (Fig. 2) to identify the metabolite being measured.) We have also measured the levels of 13,14-dihydro-15-ketocycle (Fig. 4). These values menstrual could not be correlated with ovulation (as measured by basal body temperature). Patients C and D were taking Norinyl (1+80 21-day)
PGF2, throughout the
and Gvral pills. Many of the metabolic products of PGF~~ found in urine have been identified (16,17).
Several of them (Fig. 5) have the
requisite structure (the four antigen subsites) for cross reaction with anti-13,14-dihydro-15-keto-PGF2c (the ga, lla-dihydroxy15-keto of the C-20 PG, the 7a, ga-dihydroxy-lj-keto of the C-18 and c-1.6 metabolites, and the 5a, 7a-dihydroxy-ll-keto of the C-14 metabolite).
Even some of the PGE metabolites could
cross react with this antiserum but to a lesser degree.
we
have analyzed several human urines, unextracted, for reaction The reactions
with antibodies to 13,14-dihydro-15-keto-PGF2a.
of 10 1_~1 of unextracted human urine with antisera to PGF2,, PGDl, 15-keto-PGF2a, and 13,14-dihydro-15-keto-PGF2a are shown in At the 10 ~1 level of urine, only the anti-13,14-
Table I.
dihydro-15-keto-PGF2c cross reacts significantly with metabolites present in urine. we can assign numbers for this serologic activity with anti-13,14-dihydro-15_keto-PGF2at if we assume that the c-14, c-16, and C-18 metabolites (Fig. 5) react as effectively as the C-20 metabolite. This estimate probably represents a minimal level of PGF production not Only on the basis of the above assumption but also because some PGF metabolites present do not have the requisite four subsites and therefore do not cross react.
792
JUNE 1973
VOL. 3 NO. 6
PROSTAGLANDINS
13,14-Dihydro-
I5-Kcto
I5-Kefo-PGF2,
-PGFza
PGF2.
.
IO
9
Fig. and
Levels
3.
seru-n,
represent
'-nean . The are
JUNE
of
in hulaan
DGE',,
also
1973
1~,14-dihydro-15-keto-PGF2a, sera.
the
mean
The
values,
and
the
lj,l4-dihydro-15-keto-PGF~~
15-lceto-PGFza,
expressed
standard _
as
ng/ml
deviation
levels
of
each
of
the
subject
;.ncluded.
VOL.
3 NO. 6
793
PROSTAGLANDINS
Fig. wonen
4.
pressed
794
Levels
throughout as ny/ml
of lj,14-dihydro-13-l;eto-PG1~LU the ,nenstrual cycle.
in sera of
The values
are ex-
serum.
JUNE 1973
VOL. 3 NO. 6
PROSTAGLANDINS
Fig.
5.
PGF2cz
metabolites
subjects
(16,17).
subsites
recognized
JUNE 1973
Enclosed
identified
in urine of female
areas contain
the four antigenic
by anti-13,14-dihydro-15_keto-PGF2a.
VOL. 3 NO. 6
795
PROSTAGLANDINS
The levels of PGF metabolite(s) males and 10 females
expressed
creatinine
in Table
duction
are given
of PGF/24
in the urines
of 11
both as 114124 hr and wg/g of II.
The minimal
hr would be around
value
for pro-
663-148 ILg for males and
j5-88 pg for females. We have also determined
the levels of metabolites
in rat
urines
that cross react with anti-lj,l4-dihydro-lj-keto-PGF
It was
found that in most rat urine
olites
that cross react with anti-lj,14-dihydro-15-keto-PGF and anti-PGBl
anti-15-keto-PGF2c,
All urines
(Table III).
reacted
samples
5 ~1 contained
but not with extensively
.
2a metab2aJ
anti-PGlr'2,(X
with
anti-lj,l4-
dihydro-15-keto-PGF2a
at the 10 1~1 level, but at tllis Level,
there was significant
cross reaction
with unextracted Table
rat urine)
IV are calculations
The daily production
from this inhibition as m
expressed
with respect
excretion,
The younger
PGF than man.
with
PGF metabolite/g
of
of PGF in the rat is much
less than in man, but when to creatinine
non-specific The data in
with anti-PGF2c.
obtained
5 ~1 of rat urine and are expressed creatinine.
(possibly
as a specific
activity
the rat produces
rats produce
more
more PGP than older
rats. DISCUSSION BecauSe
of the large number
quantitative measured bodies
and qualitative
groups
with
specificity
PGF compounds
is available.
toward
15-keto-PGF2o,
analysis
groups
antibodies
on
functional will
antibodies among PGFl,,
and 13,14-dihydro-
antibodies between
as
for some of the
distinguish
13,14-dihydro-15-keto-PGF20,
we have prepared
fluids,
by these procedures
of antibodies
easily
structures,
of anti-
functional
we have prepared
that do not distinguish
and PGF 2o (14).
on a battery
different
(1.5). We also have prepared
hydro-PGF20
796
PGF2, which
related
of biological
if these different
Such a library
be simplified.
PGF2,
toward
Of course,
are immunodominant,
directed
depend
by radioimmunoassay,
the PG molecules.
of closely
analyses
to lj,l4-di13,14-dihydro-PGF2a to PGFla
JUNE 1973
(PGFl,
VOL. 3 NO. 6
PROSTAGLANDINS
reacts
more effectively
than PGF2,).
bodies
to 15-keto-PGF2c
that react much more effectively
15-keto-PGF2c here,
than 13,14-dihydro-PGF2c
antibodies
tinguish
between With
PGF2,.
to 13,14-dihydro-l5-keto-PGF2c 15-keto-PGF2c
and identify
sample.
of different different
and PGF2,.
specificities,
compounds
with
As reported
do not dis-
we can begin
all of these PG compounds
Without
anti-
and 13,14-dihydro-15-keto-
the use of all of these antisera,
to guantitate biological
We have prepared
such a catalogue some physical
must be used prior
in a
of antibodies
separation
of the
to assay with a single
antiserum. As measured
by radioimmunoassay
with
in human sera have been measured. PGF2c metabolite, 13,14-dihydro-15-keto-PGF2,_, higher
than those of the metabolite,
This conclusion leagues
had been reached
(l2,l3), who quantitated
metabolite,
reported
analysis
by Samuelsson
than the administered
13, lb-wdro-15-keto-PGF2c than endogenous levels of PGF2,.
as measured
sera were reported average greater,
PGF2,.
4.8
ng/ml
(Fig. 3),
They
were
20-
Endogenous
be expected
levels of
to be higher
The only reported
endogenous
in serum is O-240 pg/ml although
less than 0.5
ng/ml
normal
in endogenous
in one
six other
(12).
Our
is considerably
and we even have observed
in a presumably
this discrepancy
and its
by mass
of PGF2,.
level of 13,14-dihydro-15-keto-PGF2c
as high as 26 ng/ml Despite
and his col-
in plasma
by radioimmunoassay, to have
and PGF2,.
the levels of PGF2,
also would
serum,
are significantly
after administration
for this major metabolite
and
15-keto-PGF2c,
that the levels of the latter metabolite
30fold higher
value
antisera,
The levels of the
13,14-dihydro-15-keto-PGF2c,
spectrometric
several
13,14-dihydro-15-keto-PGF2c
levels of 15-keto-PGF2c,
serum
levels
a value
(Fig. 4~).
in blood
of
13,14-dihydro-15-keto-PGF2c,
the levels of 13,14-dihydro-15-keto-
PGF2,
much better
reflect
of PGF2,
PGF production
or the initial
surprising,
JUNE 1973
metabolite,
since many tissues
VOL. 3 NO. 6
than do the blood
15-keto-PGF2o.
have high
15-hydroxy
This
levels
is not
PG dehydro-
797
PROSTAGLANDINS
genase
and 15-keto-PG-A13,14
With
reductase
such high concentrations
activities
(18).
ofQ,l&dihydro-15-keto-
in sera, could the PGF2, levels previously obtained PGF2a radioimmunoassay merely reflect cross reaction of 13,14with anti-PGF20?
dihydro-15-keto-PGF20
used in our laboratory, reacts
average
the anti-PGF20
13,14-dihydro-15-keto-PGF2c
Our PGF2,
less than l$.
er (6-l?“&) .
With
however,
suggests
level of 500 pg of 15-keto-PGF2o
13,14-dihydro-15-keto-PGF2o
cross
levels are considerably
The same reasoning,
average
(about g$ of the
level) reflects,
of the 13,14-dihydro-15-keto-PGF2o
anti-15-keto-PGF2o
(cross reaction
is 5$).
data are not sufficiently to calculate
of 15-keto-PGF2c
13,14-dihydro-15-keto-PGF2c
a constant
mass spectrometric production culated
have
(19).
istration
as measured
led to the assignment daily rates were
We have determined
of [3~]~~~2.
urinary
metabolites
PGF2o.
These
tion obtained
on analyses
with antiserum
calculations with
after admin-
a minimal
produc-
urine after demonstraurine did not inhibit
(Table I).
The daily produc-
from mass spectrometric
150-330
kg in males
data of the major urinary
metabolite,
were
and 20-40 bg in females.
our minimal
daily production
PGF, determined
66-148
spectrometry cedures
798
from the serologic
activities
of urine
kg in males and 35-88 pg in females.
tive analysis
of blood
of
made from the per cent inhibi-
tion rates of PGEl and PGE2, calculated
were
of
cal-
to 13,14-dihydro-15-keto-
of unextracted
non-specifically
by
of the cross reactions
10 ~1 of unextracted
tion that this quantity immune binding
were
ratio of
in each serum.
in urine,
These
our
at the low levels
from the yield of the major metabolites
tion rate for PGF based
with
or variable
to 15-keto-PGF2,
analysis,
rates to PGE
in part,
Unfortunately,
accurate
The levels of major metabolite
great-
that our
the cross reaction
analytical
by
samples,
As in compara-
levels of PG by radioimmunoassay
(20), the levels obtained
rates of
and mass
by the immunologic
pro-
are higher.
JUNE 1973
VOL. 3 NO. 6
PROSTAGLANDINS
REFERENCES 1.
Jaffe, B. M., J. W. Smith, W. T. Newton, and C. W. Parker. Radioimmunoassay for Prostaglandins. Science 171: 494, 1971.
2.
Stylos, W. A., and B. Rivets. Preparation of Specific Antiserum to Prostaglandin A. Prostaglandins 2: 103, 1972.
3.
Zusman, R. M., B. V. Caldwell, L. Speroff, and H. R. Behrman. Radioimmunoassay of the A Prostaglandins. Prostaglandins 2: 41, 1972.
4.
Levine, L., and H. Van Vunakis. Antigenic Activity of Prostaglandins. Biochem. Biophys. Res. Commun. 41: 1171, 1970.
5.
Levine, L., R. M. Gutierrez-Cernosek, and H. Van Vunakis. Specificities of Prostaglandins Bl, F AntigenAntibody Reactions. J. Biol. Chem. 24@t:a$8F2$5971.
,0.
Caldwell, B. V., S. Burstein, W. A. Brock, and L. Speroff. Radioimmunoassay of the F Prostaglandins. J. Clin. Endocr. 33: 171, 1971.
7.
Kirton, K. T., J. C. Cornette, and K. L. Barr. Characterization of Antibody to Prostaglandin F20. Biochem. Biophys. Res. Commun. 47: 903, 1972.
8.
Orczyk, G. P., and H. B. Behrman. Ovulation Blockade by Aspirin or Indomethacin. In vivo evidence for a Role of Prostaglandin in Gonadotrosii??&retions. Prostaglandins 1: 3, 1972.
9.
Gutierrez Cernosek, R. M., L. M. Morrill, and L. Levine. Levels in Peripheral Sera of Man. Prostaglandin F Prostaglandins S? 71, 1972.
10. Yu, s. c., and G. Burke. Antigenic Activity of Prostaglandins: Specificities of Prostaglandins E , A and F2o AntigenAntibody Reactions. Prostaglandins 2: $1, 1972. 11. Levine, L., and R. M. Gutierrez-Cernosek. Preparation and Specificity of Antibodies to 15-keto-prostaglandin F20. Prostaglandins 2: 281, 1972. .. 12. Granstrom, E., and B. Samuelsson. Development and Mass Spectrometric Evaluation of a Radioimmunoassay for 9a, lladihydroxy-15-ketoprost-5-enoic Acid. FEBS Letters 26: 211, 1972. 13. Samuelsson, B. Endogenous Synthesis of Prostaglandins. Third Conference on Prostaglandins in Fertility Control, WHO Research Institutet, Stockholm, 1972, p. 189.
JUNE 1973 VOL. 3 NO. 6
799
PROSTAGLANDINS
14.
Levine, L. Antibodies to Pharmacologically Active Molecules: Specificities and Some Applications of AntiProstaglandins. Pharmacological Reviews, in press.
15.
Gutierrez-Cernosek, R. M., and L. Levine. Serological Aspects of Prostaglandins. J. Reproductive Med., in press.
16.
Granstrgm, E., and B. Samuelsson. On the Metabolism of in Female Subjects. II. Structures of ;yOs;~~;;~;;:,F,ea . J. Biol. Chem. 246: 7470, 1971. ,I Granstrom, E. on the Metabolism of Prostaglandin F in E'emaleSubjects. Structures of Two Cl4 Metabolites?a sur. J. Biochem. 25: 581, 1972.
17.
18.
Samuelsson, B., E. Granstrgm, K. Green, and M. Hamberg. Metabolism of Prostaglandins. Ann. N. Y. Acad. Sci. 180: 138, 1971.
19.
Hamberg, M. Inhibition of Prostaglandin Synthesis in Man. Biochem. Biophys. Res. Commun. 49: 720, 1972.
20.
Sanuelsson, B., Convener. Round Table Discussion. vances in Biosciences 9: 1973, in press.
800
JUNE 1973
Ad-
VOL. 3 NO. 6
PROSTAGLANDINS
Table I. Inhibition of Prostaglandin Antiprostaglandin Reaction by 10 ~1 Human Urine
sub-
ject 1 2
3 4 5 6 7 8
9 10 11 12
l3 14 l5 16 17 18
19
Anti-13,14dihydrol+ketoPGF2, (S) 21
l9 15 29 17 25 9 20 18 11
17 l9 l5 l5 l9 24
29 13 10
JUNE 1973 VOL. 3 NO. 6
Anti-l+ keto-PGF2a ($1
6 6 0 5 0 0
AntiPGF2, -@J-m 1
7 3 3 0 0
1
AntiPGBl 7
5 3 1 7 3 0
0 0 0 0 0
0 0 0
3
1
0 0 0 0
5 6 0
2
2
0
5
3
2 2
3 7 1 0 4
4
0
0
3
0
4
0
3
801
PROSTAGLANDINS
Table If.
Levels
of 13,1&Dihydro-15-Keto-Prostaglandin
Metabolites*
in Human Urine Females
Males Subject
yg/24 hr
cLg/g creatinine
Subject
kg/24 hr
clg/g creatinine
1
147
85
1
88
2
107
52
2
80
58 68
3
121
55
4
66
38
3 4
85 45
58 41
5
80
65
5
35
26
6
82
45
6
59
43
7
78 89
35 41
7 8
40 50
37 52
9 10
70 52
50 5a
-
-
8
9
86
44
10
126
64
11
148
35
49 I 12 60 " 19 103 + 29 52 I 15 Mean + S.D. *., Metabolic products that cross react with anti-lj,l&dihydrol+keto-PGF2a.
802
JUNE 1973
VOL. 3 NO. 6
PRO.STAGLANDIN.9
Table
III.
Inhibition
Prostaglandin
Anti-13,14dihydro-15keto-PGF2a
Rat
+* 8
of Prostaglandin-Anti-
Reactions
by
5 ~1 of Rat Urine
Anti-l+ keto-PGF2c
AntiPGF2cl 03 0
($1
($1
35
13
40
17
36
15
5 0
34 33
13 12
0
35
12
35
15
30
1
AntiPGBl -7K 4 5 N.D.* 5 1 5
3 6
17
3
7
9
23
1-5 11
5
5
10
28
12
0
5
11
31 16
14
5 2
11
0
13 14
16
7 6
31
11
2
3 10
15 16
30
13
2
12
17
7
5
4
12
2
*
** 1 ~1 of this rat N.D. = not determined. urine was used in the assay reaction mixture.
JUNE
1973
VOL.
3 NO. 6
803
PROSTAGLANDINS
Table
Iv.
Levels of 13,14-Dihydro-15-Keto-
Prostaglandin
Metabolites
in Urine
of Female Rats Rat
1-LgPG/g creatinine 354
122
241 520
204 224
421
250
347
139
425
271
342
203
284
804
kg PG/g creatinine
(250
7xEq
Mean +, S.D.
Rat
367 +_ 88
125 192 “_ 57
JUNE 1973
VOL. 3 NO. 6