From the Department of Environmental Health Sciences
of the ** Istituto di Ricerche Farmacologiche "Mario Negri," Via
Eritrea 62, 20157 Milano, Italy and the
Faculty of Pharmacy,
UPRES A CNRS 5074, UM I, F-34060 Montpellier, France
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ABSTRACT |
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F2-isoprostanes are
prostaglandin-like compounds derived from nonenzymatic free
radical-catalyzed peroxidation of arachidonic acid.
8-epi-Prostaglandin (PG) F2 F2-isoprostanes are a complex family of isomeric
F2-prostaglandin
(PG)-like1 compounds derived
from free radical-catalyzed nonenzymatic peroxidation of arachidonic
acid (1). These compounds are currently used as novel biomarkers of
lipid peroxidation in vivo (2-4).
F2-isoprostanes esterified to tissue or plasma lipids or in
the free form in body fluids have in fact proved to be useful indices
of local or systemic oxidant stress (2-4). Assay of selected major
isomers such as 8-epi-PGF2 In view of further refining the quantitative assessment of the
endogenous formation of F2-isoprostanes, it would be
important to investigate their degradation in vivo. The
identification of the major metabolites of selected
F2-isoprostanes would be useful for two main reasons.
First, quantitation of major metabolites in addition to the parent
compound may allow a more accurate evaluation of the overall production
of the biomarker in vivo while adding significance to
individual measurements. Second, identification of
F2-isoprostane metabolites may help finding circulating
compounds that can be measured without the risk of artifactual
production ex vivo (2, 4).
Theoretically, metabolism of F2-isoprostanes may proceed as
for F2-prostaglandins (6-8), i.e. they may
undergo various combinations of To date, two distinct F2-isoprostane isomers,
8-epi-PGF2 Materials--
8-epi-PGF2 Immunoaffinity Chromatography--
IAC extractions were
performed as described previously, using immunosorbents prepared with
antibodies raised against 8-epi-PGF2 Derivatization--
Samples were converted to various
derivatives before GC-MS analysis. Pentafluorobenzyl (PFB) ester and
trimethylsilyl (TMS) ethers were prepared as described (14),
tert-butyldimethylsilyl (tBDMS) ethers by treating dried
samples with 10 µl of
N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA, Pierce, Rockford, IL) plus dry pyridine (10 µl) and acetonitrile (30 µl) for 1 h at 60 °C, cyclic butyl boronate
derivatives (BB) by adding 1-butane boronic acid (60 µl of a 0.3 mg/ml solution in acetonitrile) for 10 min at 40 °C, and methyl
esters (ME) by addition of ethereal diazomethane (150 µl) at room temperature.
GC-MS--
GC-negative ion chemical ionization (NICI)-MS was
performed as described (16) using a Finnigan 4000 mass spectrometer.
Briefly, GC operating conditions were: NB-54 (5% phenyl, 95%
dimethylpolysiloxane) or CP-Sil 19 CB (14% cyanopropylphenyl, 86%
dimethylpolysiloxane) fused silica capillary columns (length, 25 m; inner diameter, 0.32 mm; film thickness, 0.12 µm); solvent-split
injection (50 to 300 °C); oven temperature, isothermal at 120 °C
for 1 min, and then programmed to 300 °C at 25°/min; helium was
used as a carrier gas. NICI operating conditions were: selected ion
recording (SIR) of carboxylate anions (M-181, loss of
·CH2C6F5); full scan mode:
100-850 m/z; methane as reagent gas; electron
energy, 100 eV. GC-electron impact (EI)-MS was performed using a VARIAN
SATURN 2000 Ion Trap mass spectrometer. GC operating conditions were:
HP-5 MS (5% phenyl, 95% dimethylpolysiloxane) fused-silica capillary
column (length, 30 m; inner diameter, 0.25 mm; film thickness,
0.25 µm); splitless injection (240 °C); oven temperature,
isothermal at 160 °C for 1 min, and then programmed to 310 °C at
20°/min; helium as carrier gas. MS conditions were: ion trap
temperature, 200 °C; mass range, 50-650 m/z;
electron energy, 70 eV.
Metabolite Quantitation by IAC-GC-NICI-MS--
To control IAC
extraction efficiency for metabolite quantitation without stable
isotope-labeled analogues, we devised the following procedure:
(a) test urine samples containing different pre-determined
amounts of endogenous 8-epi-PGF2 Chemical Synthesis of
ent-2,3-Dinor-5,6-dihydro-8-epi-PGF2 C Values--
A mixture of normal saturated C18-C24 fatty acids
were derivatized to PFB esters and analyzed by GC-NICI-MS in the SIR
mode, monitoring their carboxylate anions (M-PFB). Plots of retention times (y) with two different GC columns against carbon
number (x) (r = 0.999) were used to
calculate equivalent C values from the retention times of
different PFB derivatives (TMS, BB-TMS, or tBDMS) of each metabolite
(Table I). The same procedure was used to calculate C values
of ME-TMS metabolites with C18-C24 fatty acid methyl esters (see
`Results").
Human Urine--
6-h urine was collected from healthy volunteers
(8 males, 2 females; age, 24-57 years; 5 smokers and 5 nonsmokers).
Four nonsmokers were given two oral doses of naproxen sodium (550 mg at
12-h interval), and urine was collected before treatment and after the
second dose. Samples were stored at Rat Urine--
24-h urine was collected from male Crl:CD (SD)BR
rats (300-350 g) kept in metabolic cages.
8-epi-PGF2 Isolated Rat Hepatocytes--
Hepatocytes were isolated from fed
male Crl:CD (SD)BR rats (230-260 g) by perfusing the liver with a
collagenase solution (collagenase type IV, Sigma) by the method of
Seglen (18). After removing the liver capsule, the cell suspension was
passed through gauze. The hepatocytes were washed three times (50 g × 1 min) and suspended in Leibovitz L-15 medium (Life
Technologies, Inc., Paisley, Scotland) supplemented with 18 mM Hepes, 1 µg/ml insulin, 50 µg/ml gentamicin, and 5%
fetal calf serum (Life Technologies, Inc.) at a cell density of 1 × 106 cells/ml. The viability, assessed by trypan blue
exclusion, was higher than 85%. After 3 h of adhesion the medium
was changed with an equal medium without fetal calf serum and
gentamicin, and the cells were incubated for 20 min with and without
8-epi-PGF2 Search Strategy for Identification of Endogenous Urinary
Metabolites--
Structural identification of immunoextracted urinary
metabolites was achieved by different complementary approaches, all
suggesting that they were the following:
2,3-dinor-8-epi-PGF2 Selected Ion Recording of Different
Derivatives--
Different portions of an IAC extract of human urine
(40 ml on immunosorbent C) were derivatized to PFB-TMS, PFB-tBDMS, or PFB-BB-TMS and analyzed by GC-NICI-MS in the SIR mode. Carboxylate anion m/z values to be monitored were calculated
for the different derivatives and for each of the following putative
structures: trihydroxy-dinor-prostadienoic acid (C18:2),
trihydroxy-dinor-prostaenoic acid (C18:1), and
trihydroxy-tetranor-prostaenoic acid (C16:1). For each derivative,
peaks were registered at the expected m/z value
with C values changing coordinately with the authentic
parent compounds on the different columns (Table I), thus confirming the proposed structures. Additional structural information was obtained
by the formation of butylboronate cyclic derivatives (PFB-BB-TMS),
implying that the two 9,11-hydroxyls are in the cis position
for all compounds (11). Data regarding the C16 metabolite of
8-epi-PGF2 Full Scan Mass Spectrometry--
Full scan NICI mass spectra were
obtained from a 35-ml human urine sample extracted on immunosorbent C
and derivatized as PFB-TMS. The mass spectra of all metabolites
revealed a carboxylate anion isotopic cluster (M-181, loss of
pentafluorobenzyl group) as the only prominent peak, with the expected
m/z values (Table I) and relative isotopic
abundance (data not shown). To obtain additional evidence of the
identity of urinary 2,3-dinor-8-epi-PGF2 Rat Urine--
Aliquots of pooled rat urine (10 ml) were also
extracted on immunosorbent C and processed as described above for
NICI-MS analysis. The same compounds were identified as for human
urine, with the exception of the C18:2 derivative of
PGF2 Identification of Exogenous Hepatocyte Metabolites--
To confirm
the identity of the urinary metabolites we verified that their gas
chromatographic and mass spectral characteristics were identical to
those of Urinary Metabolite I versus Hepatocyte-derived
2,3-Dinor-8-epi-PGF2 Selectivity of Immunoextraction--
The three
anti-8-epi-PGF2 Endogenous Levels in Human Urine--
Endogenous
8-epi-PGF2
Naproxen given to healthy nonsmokers (n = 4) did not
alter the urinary excretion of the three compounds (pre-
versus post-treatment values:
8-epi-PGF2 Free radical-mediated nonenzymatic peroxidation of membrane-bound
arachidonic acid results in the formation of
F2-isoprostanes, a complex family of 64 compounds isomeric
to PGF2 Assay of urinary F2-isoprostanes and in particular of
distinct major isomers such as 8-epi-PGF2 To date, a single major metabolite of
8-epi-PGF2 Using a different approach aimed at revealing endogenous metabolites
normally present in urine, we have now shown that at least two major
metabolites of 8-epi-PGF2 A possible explanation for
2,3-dinor-8-epi-PGF2 The collective evidence that Metabolite I found in urine is the
2,3-dinor metabolite of 8-epi-PGF2 We have identified another Profile measurements of 8-epi-PGF2 Treatment of volunteers with a dose of naproxen significantly
inhibiting cyclooxygenase activity in vivo (23) did not
reduce excretion of either 8-epi-PGF2 In conclusion, we have identified and measured two major endogenous
degradation products of 8-epi-PGF2, a major
component of the F2-isoprostane family, can be conveniently
measured in urine to assess noninvasively lipid peroxidation in
vivo. Measurement of major metabolites of endogenous
8-epi-PGF2
, in addition to the parent
compound, may be useful to better define its formation in
vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2
is the only identified metabolite of
8-epi-PGF2
in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another
major endogenous metabolite,
2,3-dinor-8-epi-PGF2
, in human and rat
urine. The identity of these compounds, present at the pg/ml level in
urine, was proven by a number of complementary approaches, based on:
(a) immunoaffinity chromatography for selective extraction;
(b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated
rat hepatocytes; and (d) chemical synthesis of the
enantiomer of
2,3-dinor-5,6-dihydro-8-epi-PGF2
as a
reference standard. In humans, the urinary excretion rate of both dinor
metabolites is comparable with that of
8-epi-PGF2
. Both metabolites increase in
parallel with the parent compound in cigarette smokers, and they are
not reduced during cyclooxygenase inhibition. Another
-oxidation
product, 2,3,4,5-tetranor-8-epi-PGF2
, was
identified as a major product of rat hepatocyte metabolism. In
conclusion, at least two major
-oxidation products of
8-epi-PGF2
are present in urine, which may
be considered as additional analytical targets to evaluate
8-epi-PGF2
formation and degradation in vivo.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
or isoprostane
F2
-I in human urine has proven an accurate noninvasive
means of evaluating their formation in vivo (3, 5).
-oxidation, double bond reduction,
alcohol group oxidation,
-hydroxylation, and
-oxidation, which
per se may lead to a miriad of metabolites. The structural
differences between the four regioisomers and the stereochemical
differences within each regioisomer class (1) will likely cause major
differences in the way each product is degraded, further complicating
the picture. Therefore, metabolism of F2-isoprostanes
cannot be studied with a single parent compound as a model nor can a
single type of metabolite be chosen to monitor degradation of
F2-isoprostanes as a group. Moreover, administration of
relevant amounts of a single isomer to study its catabolism may
somewhat alter its metabolic pathway, possibly favoring reactions that
are normally inhibited by the presence of other competing endogenous
substrates. Therefore, a reasonable approach to characterize F2-isoprostane catabolism would be to identify the major
endogenous metabolites of the principal isomers produced in
vivo.
and isoprostane
F2
-I (5, 9), have been positively identified in
vivo. Metabolism of
[3H]8-epi-PGF2
has been studied
by Roberts et al. (10) in humans, and a single compound,
2,3-dinor-5,6-dihydro-8-epi-PGF2
, has been
identified as the major urinary metabolite. The same group identified
other F2-isoprostane metabolites as a group of C16 dioic
compounds of unknown stereochemistry, with two hydroxyl groups, one
keto group, and one double bond (11). We report here the identification
of two endogenous urinary metabolites of
8-epi-PGF2
by a strategy taking advantage of
immobilized antibodies to capture endogenous cross-reactants for
subsequent identification/measurement by gas chromatography-mass
spectrometry (12, 13).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and
3,3',4,4'-[2H4]-8-epi-PGF2
were purchased from Cayman Chemicals (Ann Arbor, MI).
PGF2
and
3,3',4,4'-[2H4]-PGF2
were from
Upjohn Co. (Kalamazoo, MI).
ent-12-epi-PGF2
(15S
and 15R), 12-epi-PGF2
(15S and 15R), and
15R-8-epi-PGF2
, were synthesized
in our laboratory as described previously (14, 15).
(16).
Briefly, urine was diluted (1:2-1:5) with 50 mM phosphate buffer (pH 7.4) spiked with 2H4-labeled
internal standard, if required, and percolated slowly through a IAC
column prepared with the immunosorbent (IgG fraction of the antiserum
coupled to CNBr-activated Sepharose 4B). After washing with distilled
water, the column was eluted with acetone:water (95:5, v/v), and the
eluate was dried under an air stream.
were
percolated in succession through three IAC columns without
internal standard; (b) each column was then eluted
separately in tubes containing a fixed amount of the internal standard
2H4-8-epi-PGF2
;
(c) GC-NICI-MS quantitation of the metabolites recovered in
the three IAC eluates from the same urine sample showed that all
compounds had been extracted almost completely in the first two
rounds (96-100% for 8-epi-PGF2
and
2,3-dinor-8-epi-PGF2
, 87-92% for
2,3-dinor-5,6-dihydro-8-epi-PGF2
). Two-round
extractions were therefore performed for unknown samples, the total
amount of each metabolite being calculated by summing the two partial results obtained. Quantitation was done by using standard plots of
8-epi-PGF2
/2H4-8-epi-PGF2
peak area ratio (y) versus the amount of
8-epi-PGF2
(x) (r = 0.9999), assuming that equimolar amounts of the C20 and C18 analogues
give similar carboxylate anion peak area response, because this ion
carries most of the total ion current.
--
The total
synthesis of the enantiomer of
(15S)-2,3-dinor-5,6-dihydro-8-epi-PGF2
was carried out as we have described recently (17) using
diacetone-D-glucose as starting material. After methylation
of the carboxyl acid with diazomethane, the 15R-epimer
(ent-2,3-dinor-5,6-dihydro-8-epi-PGF2
)
could be separated from the 15S-epimer by flash
chromatography over silical gel. Relative stereochemical assignments
were made by NOE 1H NMR study.
20 °C.
(10 µg dissolved in 0.5 ml of
sterile phosphate-buffered saline) was injected intravenously into a
rat whose urine was collected for 24 h before and after treatment.
Samples were kept at
20 °C until analyzed. Procedures involving
animals and their care were conducted in conformity with the
institutional guidelines that are in compliance with national2 and international
laws and policies.3
or PGF2
. The
cell-free incubation medium was collected and stored at
20 °C
until analyzed. Metabolites were extracted both by IAC and by C18 solid
phase extraction (SPE). The latter was performed by loading the sample
at pH 3.5, washing with water and petroleum ether, and then eluting
with methyl formate. Aliquots of IAC and C18 SPE extracts were dried
and derivatized to ME-TMS or PFB-TMS and analyzed by GC-EI-MS or
GC-NICI-MS, respectively.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Oxidation Metabolites of Endogenous
8-epi-PGF2
--
Because polyclonal antibodies raised
against prostanoids coupled to the carrier protein via the carboxyl
group often cross-react with the corresponding
-oxidation products
(12, 13, 19), we tested three antibodies (A, B, and C) raised against
8-epi-PGF2
(16) for their ability to extract
any of its possible
-chain-shortened (C18 and C16) metabolites.
Aliquots (10 ml) of a human urine pool were extracted using three
different immunosorbents prepared with A, B, and C antibodies. Extracts
were derivatized as PFB-TMS and first analyzed by GC-NICI-MS in the SIR
mode, monitoring the carboxylate anions of
8-epi-PGF2
(m/z 569)
and those of its putative C18 and C16 homologues
(m/z 541 for C18:2, m/z 543 for C18:1, and m/z 515 for C16:1). The three
immunosorbents showed different selectivity toward the C18 and C16
metabolites, with A extracting only
8-epi-PGF2
and no metabolites and B
extracting 8-epi-PGF2
and a single C18:2
metabolite (I), whereas C extracted
8-epi-PGF2
plus PGF2
and
doublets of their chain-shortened homologues C18:2 (I and Ia) and C18:1
(II and IIa) as well as a single C16 metabolite (IIIa) (Fig.
1). The GC behavior of these compounds preliminarily suggested that I and II were metabolites of
8-epi-PGF2
, whereas Ia, IIa, and IIIa were
derived from PGF2
(Table
I).
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Fig. 1.
GC-NICI-MS selected ion recording tracing of
human urine extracted by immunoaffinity chromatography (immunosorbent
C). The ions monitored for 8-epi-PGF2
and PGF2
and their metabolites are the carboxylate
anions (M-PFB) of the PFB-TMS derivatives. Peaks labeled as I and Ia at
m/z 543 are from the isotopic cluster of the
carboxylate anions recorded at m/z 541 (metabolites I and Ia). The relative amounts of the compounds cannot be
directly evaluated from this tracing, because their recovery is
different.
NICI mass spectral data and equivalent C values of different PFB
derivatives of 8-epi-PGF2, PGF2
, and their
metabolites
(I) and 2,3-dinor-5,6-dihydro-8-epi-PGF2
(II), plus
their PGF2
-derived counterparts
2,3-dinor-PGF2
(Ia) and
2,3-dinor-5,6-dihydro-PGF2
(IIa), and
2,3,4,5-tetranor-PGF2
(IIIa).
identified in rat hepatocyte
preparation (see below) are also shown in Table I. The C
values of the C18 and C16 metabolites were about 1.5 and 3 units
smaller than those of the respective parent compound for the different
derivatives, as reported for two- and four-carbon
-chain shortening
of F series prostaglandins (8, 20).
5 double bond
saturation resulted in the expected small increase in C
value for all PFB derivatives.
, full scan EI-MS analysis was performed on a IAC extract (immunosorbent C) from a 160-ml human urine sample derivatized as ME-TMS. At the
retention time of authentic
ent-2,3-dinor-5,6-dihydro-8-epi-PGF2
(C value 21.69; cf. C value for
8-epi-PGF2
: 23.18), we recorded a mixed mass
spectrum clearly showing doublets of major fragment ions at
m/z 305-307, 376-378, 395-397, and 466-468,
corresponding to the major fragment ions retaining the
-chain of
2,3-dinor-8-epi-PGF2
(as seen in the
hepatocyte preparation, Fig.
2A and Table
II) and
2,3-dinor-5,6-dihydro-8-epi-PGF2
(as seen
with the authentic enantiomer, Fig. 2D and Table II). The
difference of 2 Da in the fragment ion doublets is due to the presence
or absence of the
5 double bond. The two compounds
coeluted when analyzed as ME-TMS in these conditions. We then prepared
a similar sample but before derivatization reextracted it on
immunosorbent B, thus eliminating 2,3-dinor-5,6-dihydro-8-epi-PGF2
. We thus
obtained a mass spectrum (Fig. 2C) of weak intensity but
with all prominent ions corresponding to those of
2,3-dinor-8-epi-PGF2
(Fig. 2A and Table II).
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Fig. 2.
EI mass spectra of ME-TMS derivatives of
2,3-dinor-8-epi-PGF2 (panel A)
and 2,3,4,5-tetranor-8-epi-PGF2
(panel
B) from isolated rat hepatocytes incubated with
8-epi-PGF2
(extracted by C18 SPE),
endogenous 2,3-dinor-8-epi-PGF2
from human
urine (sequentially extracted on immunosorbents C and B) (panel
C), and chemically synthesized
ent-2,3-dinor-5,6-dihydro-8-epi-PGF2
(panel D).
EI-MS fragmentation pattern of ME-TMS derivatives of
8-epi-PGF2 and its metabolites
-chain:C1-C7 for
8-epi-PGF2
, C1-C5 for I and II, and
C1-C3 for III; b, C9-C10 or C10-C11 for
8-epi-PGF2
, C7-C8 or C8-C9 for I and II,
and C5-C6 or C6-C7 for III; c,
-chain: C13-C20
for 8-epi-PGF2
, C11-C18 for I and II, and
C9-C16 for III; d, C15-C20 for
8-epi-PGF2
, C13-C18 for I and II, and
C11-C16 for III.
(2,3-dinor-PGF2
), which was not
detectable (data not shown). In a rat treated with 10 µg of
intravenous 8-epi-PGF2
, 24-h excretion of
8-epi-PGF2
, 2,3-dinor-8-epi-PGF2
, and
2,3-dinor-5,6-dihydro-8-epi-PGF2
increased by
5.6-, 9.1-, and 9.2-fold over the respective pre-treatment levels
(1.16, 1.20, and 1.55 ng/24 h), suggesting that the latter compounds
were metabolites of the former.
-oxidation products of authentic
8-epi-PGF2
or PGF2
incubated
with rat hepatocytes, which were in turn identified by EI-GC-MS as
follows. Products of 8-epi-PGF2
or
PGF2
metabolism by isolated rat hepatocytes were
extracted in parallel by IAC (immunosorbent C) and C18 SPE. Both
extracts were then analyzed by full scan EI-GC-MS. Putative
2,3-dinor-8-epi-PGF2
(Metabolite I) was
present in both extracts. Its structure was proven by the mass spectrum
of its ME-TMS derivative (Fig. 2A) showing the ions expected
in agreement with the fragmentation pattern described in Table II.
Another abundant
-oxidation metabolite was identified in this
preparation as 2,3,4,5-tetranor-8-epi-PGF2
on
the basis of the EI mass spectrum and GC behavior (C value: 20.12). Its mass spectrum (Fig. 2B) was almost identical to
that reported by Green (8) for the corresponding metabolite of
PGF2
. 2,3,4,5-Tetranor-8-epi-PGF2
could be observed
in the C18 SPE but not in the IAC extract. This result indicates that
immunosorbent C does not extract this metabolite, and therefore we
cannot presently exclude nor prove its presence in urine.
2,3-Dinor-5,6-dihydro-8-epi-PGF2
was a minor
product in the hepatocyte preparation, as judged by NICI-MS analysis of
PFB-TMS derivatives of C18 and IAC extracts. In the hepatocyte
preparation incubated with PGF2
, we confirmed the
identity of 2,3-dinor-5,6-dihydro-PGF2
and
2,3,4,5-tetranor-PGF2
but could not find
2,3-dinor-PGF2
, either in SPE or in IAC extracts
examined by both EI- and NICI-MS, similar to what had been observed in
rat urine. The same products of PGF2
metabolism were
identified by Sago et al. (21) in rat hepatocytes. The EI
mass spectra of PGF2
metabolites (not shown) were very similar to those published previously (8) and to those we have shown
here for the corresponding metabolites of
8-epi-PGF2
. This similarity was expected,
because it had been observed for the parent epimeric compounds,
8-epi-PGF2
and PGF2
(not shown).
--
The immunoaffinity behavior
and GC-MS characteristics of urinary Metabolite I versus
hepatocyte-derived 2,3-dinor-8-epi-PGF2
, taken as a reference, were compared directly as follows. Hepatocyte medium and human urine were immunoextracted in parallel on two distinct
antibodies (immunosorbents B and C) and derivatized to PFB-TMS,
PFB-tBDMS, and PFB-BB-TMS. Samples were then analyzed by GC-NICI-MS in
the SIR mode in the conditions described above, but with a lower GC
temperature programming rate (10 °C/min), resulting in retention
times of 14.45, 18.02, and 16.32 min for the PFB-TMS
(m/z 541), PFB-tBDMS (m/z
667), and PFB-BB-TMS (m/z 463) derivatives of
2,3-dinor-8-epi-PGF2
. In all these
combinations of immunoselective capture, derivative formation, GC
separation and mass-selective detection, Metabolite I behaved
identically to 2,3-dinor-8-epi-PGF2
. The
retention times of the three derivatives of Metabolite I were 14.45, 18.02, and 16.32 min. When similar amounts of
2,3-dinor-8-epi-PGF2
and Metabolite I were
co-injected, the peak area increased proportionally, whereas the width
at half-maximum remained unchanged.
immunosorbents tested in this
study selectively capture their nominal antigenic ligand from a complex
matrix such as urine (16). As shown above, only immunosorbent B and C
cross-reacted with other endogenous PGF2 compounds. We tested the stereoselectivity of immunosorbents B and C against five
available Class IV F2-isoprostane diastereoisomers, namely 15R-8-epi-PGF2
,
15S-12-epi-PGF2
,
15R-12-epi-PGF2
, 15S-ent-12-epi-PGF2
,
and
15R-ent-12-epi-PGF2
.
Together with 8-epi-PGF2
, these compounds
comprise almost the complete array (six of eight) of the Class IV
F2-isoprostanes predicted as most abundant, i.e.
those bearing the alkyl chains in the cis position. Neither
immunosorbent captured any of the compounds tested (<1% recovery). In
addition, immunosorbent C, which efficiently captures
2,3-dinor-5,6-dihydro-8-epi-PGF2
, cannot
recognize its enantiomer,
ent-2,3-dinor-5,6-dihydro-8-epi-PGF2
(<1% recovery).
and its metabolites were excreted
in comparable amounts in human urine (Fig.
3). In smokers, who excreted higher
amounts of 8-epi-PGF2
in agreement with
previous observations (16, 22),
2,3-dinor-8-epi-PGF2
and
2,3-dinor-5,6-dihydro-8-epi-PGF2
increased in
parallel with the parent compound (Fig. 3). Urinary excretion rate of
2,3-dinor-8-epi-PGF2
was highly correlated to
that of 8-epi-PGF2
in a group of five smokers
and five nonsmokers (r = 0.89, p = 0.0005). A weaker but significant correlation was observed for
2,3-dinor-5,6-dihydro-8-epi-PGF2
(r = 0.77, p = 0.01).
View larger version (12K):
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Fig. 3.
Correlation between urinary excretion of
8-epi-PGF2 and its metabolites in a group of
healthy volunteers (five nonsmokers and five smokers).
, 8.14 ± 2.05 versus 8.63 ± 1.51 ng/h; 2,3-dinor-8-epi-PGF2
, 8.95 ± 2.30 versus 9.51 ± 0.39 ng/h; 2,3-dinor-5,6-dihydro-8-epi-PGF2
, 6.37 ± 1.30 versus 7.11 ± 1.04 ng/h).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(1, 2). These products are found both esterified
to tissue and plasma lipids and in the free form in body fluids, so
that they can be used to evaluate local or systemic lipid peroxidation
in vivo (2, 3). In humans, increased
F2-isoprostane levels have been found in different
physiopathological conditions putatively associated with free
radical-mediated oxidant damage, such as atherothrombotic disease,
diabetes, and cigarette smoking (2-4).
and
isoprostane F2
-I is of special interest for human
studies, because the collection is noninvasive, the measurement is
time-integrated, and the analytes are stable and cannot be formed
artifactually ex vivo as in plasma (2-4). Although
8-epi-PGF2
may also be formed enzymatically by cyclooxygenase (3), it has been repeatedly shown that its urinary
excretion mainly reflects its free radical-mediated formation, at least
in humans (3, 23). To evaluate 8-epi-PGF2
overall formation in vivo, it would be useful to identify
and measure its major urinary metabolites. In fact, considering that
catabolism of 8-epi-PGF2
may be variable in
different subjects or under particular physiopathological conditions,
the additional assay of degradation products would improve the
significance and accuracy of these measurements. Identification of
endogenous metabolites would also be useful to define new
F2-isoprostane analytical targets for assaying lipid-rich
body fluids without the risk of overestimates due to autooxidation
artifacts ex vivo (2, 4).
, i.e.
2,3-dinor-5,6-dihydro-8-epi-PGF2
, has been
identified in urine by Roberts et al. (10). In that study,
tracer amounts of
[3H]8-epi-PGF2
were given to a
volunteer whose urine was then mixed with the urine of a monkey treated
with 500 µg of 8-epi-PGF2
. The major
radioactive human metabolites could thus be followed during urine
work-up, with enough co-migrating unlabeled products from the monkey to
allow identification by EI-GC-MS.
are excreted at a
rate similar to that of the parent compound. In addition to confirming
the presence of
2,3-dinor-5,6-dihydro-8-epi-PGF2
, we have in
fact found another
-oxidation product of similar abundance excreted
under basal conditions in humans. The same products were found in rat urine.
escaping detection in
the study of Roberts et al. (10) might lie in an incomplete
chromatographic separation from its
5 saturated
analogue. In fact, we observed that although
2,3-dinor-8-epi-PGF2
and
2,3-dinor-5,6-dihydro-8-epi-PGF2
were easily
separable by GC as PFB-TMS derivatives, they coeluted when analyzed in
similar conditions as ME-TMS derivatives. Assuming that (i) in the
experiment of Roberts et al. (10), the two compounds had
coeluted both during radioactive high pressure liquid chromatography
and during GC analysis of ME-TMS derivatives and (ii) urinary
2,3-dinor-5,6-dihydro-8-epi-PGF2
had been
much more abundant than 2,3-dinor-8-epi-PGF2
in the treated monkey, the latter metabolite might have gone undetected in the pooled human/monkey urine sample assayed by GC-MS.
can be
summarized as follows. As deducted from EI and NICI mass spectral data,
Metabolite I has the structure of a
7,9,13-trihydroxy-dinorprost-3,11-dienoic acid. Therefore it might be
any endogenous 2,3-dinor-PGF2 compound, excluding the
cyclooxygenase-derived 2,3-dinor-PGF2
, which elutes
separately in our GC conditions. Metabolite I cannot therefore derive
from a F2-isoprostane other than Class IV, i.e.
those with a 9,11,15-trihydroxy-prost-5,13-dienoic acid structure. Its
stereochemical structure is most likely that of
2,3-dinor-8-epi-PGF2
because (a)
the 2,3-dinor metabolite formed by hepatocytes from authentic 8-epi-PGF2
behaved identically to urinary
Metabolite I when analyzed by GC-NICI-MS as PFB-TMS, PFB-tBDMS, or
PFB-BB-TMS and by GC-EI-MS as ME-TMS; (b) the two distinct
anti-8-epi-PGF2
immunosorbents (B and C) used
to extract both urinary Metabolite I and hepatocyte-derived
2,3-dinor-8-epi-PGF2
do not cross-react with
several Class IV F2-isoprostane diastereoisomers; and
(c) immunosorbent C displays enantioselectivity toward
2,3-dinor-5,6-dihydro-8-epi-PGF2
. Finally,
Metabolite I increases in rat urine after administration of
8-epi-PGF2
.
-oxidation product of
8-epi-PGF2
,
2,3,4,5-tetranor-8-epi-PGF2
, in a preparation
of isolated rat hepatocytes incubated with the authentic compound, but
at this time we could not test for its presence in urine, because none
of our available anti-8-epi-PGF2
immunosorbents displayed cross-reactivity with this metabolite. The
experiment with isolated rat hepatocytes comparing metabolism of
8-epi-PGF2
and PGF2
in
identical conditions has preliminarily revealed some stereoselectivity in the
-oxidation pathway, as may be expected for compounds that are
epimers at the
-chain-bearing carbon. In fact, although a tetranor
metabolite (C16:1) was found for both of
8-epi-PGF2
and PGF2
, their
respective major C18 metabolites were
2,3-dinor-8-epi-PGF2
and
2,3-dinor-5,6-dihydro-PGF2
.
and its
metabolites have allowed us to note some interindividual variation in
metabolism, which would imply that overall formation of
8-epi-PGF2
might be estimated more accurately
with the additional monitoring of the metabolites, but this aspect has
to be specifically addressed with a broader sample. In smokers, the two
metabolites increase in parallel with the parent compound, although
correlation with 2,3-dinor-8-epi-PGF2
was
stronger than that of
2,3-dinor-5,6-dihydro-8-epi-PGF2
. Additional
observations will be needed to determine whether smokers excrete
significantly different fractions of the two metabolites. In any case,
their correlation with 8-epi-PGF2
not only
confirms their identity as degradation products but also indirectly
strengthen the hypothesis that urinary
8-epi-PGF2
mainly reflects the systemic
rather than renal formation of the compound (23).
, as we
have observed previously (23), or its metabolites. In contrast, we
observed the expected reduction of PGF2
and its assigned
metabolites (data not shown). These results further confirm the
identity of the various immunoextracted metabolites belonging to the
cyclooxygenase-dependent pathway (PGF2
series)
versus those deriving from free radical-mediated lipid
peroxidation (8-epi-PGF2
series). A
collateral finding in this study is the identification of endogenous
2,3-dinor-PGF2
in human urine, an intermediate product
that has not been detected earlier as a metabolite of exogenous
PGF2
(6, 7).
in human
and rat urine. These metabolites may be quantified in addition to their parent compound to better assess 8-epi-PGF2
formation in vivo. This study has also shown the usefulness
of exploiting cross-reactivity of immobilized antibodies for capturing
endogenous unknowns for structural analysis by GC-MS.
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ACKNOWLEDGEMENTS |
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The skillful assistance of Dr. Renzo Bagnati and Luigi Cappellini is gratefully acknowledged.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milano, Italy. Tel.: 39-02-39014497; Fax: 39-02-39001916; E-mail: chiabrando{at}irfmn.mnegri.it.
¶ Fellow of the Fondazione A. and A. Valenti.
Present address: CNR, Cellular and Molecular Pharmacology
Center, Via Vanvitelli, 32, 20129 Milano, Italy.
The abbreviations used are: PG, prostaglandin; IAC, immunoaffinity chromatography; PFB, pentafluorobenzyl; TMS, trimethylsilyl; tBDMS, tert-butyldimethylsilyl; BB, butyl boronate; ME, methyl ester; GC, gas chromatography; NICI, negative ion chemical ionization; MS, mass spectrometry; EI, electron impact; SIR, selected ion monitoring; SPE, solid phase extraction.
2 Decreto Legislativo Number 116, G.U., suppl. 40, 18 Febbraio 1992, Circolare No. 8, Gazzetta Ufficiale, 14 Luglio 1994.
3 European Economic Community Council Directives 86/609, Official Journal L 358, 1, Dec. 12, 1987; Guide for the Care and Use for Laboratory Animals, U.S. National Research Council, 1996.
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REFERENCES |
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