Plasma, urinary, and salivary 8-epi-prostaglandin F2 levels

in normotensive and preeclamptic pregnancies

Elizabeth T. McKinney, MD, Reza Shouri, MD, R. Sean Hunt, Robert A. Ahokas, PhD, and
Baha M. Sibai, MD
Memphis, Tennessee
OBJECTIVE: Our purpose was to measure and compare plasma, urinary, and salivary concentrations of
8-epi-prostaglandin F2 (8-isoprostane) in women with normotensive pregnancies and the respective concentrations in pregnancies complicated by preeclampsia.
STUDY DESIGN: Plasma, urinary, and salivary 8-isoprostane levels were measured in pregnant women with
preeclampsia (n = 40), normotensive pregnant women (n = 20), and nonpregnant women (n = 10). One-way
analysis of variance was used to determine significant differences.
RESULTS: Plasma free 8-isoprostane concentrations were increased in women with severe preeclampsia
(342 50 pg/mL), in comparison with nonpregnant women (129 17 pg/mL) and normotensive pregnant
women (150 11 pg/mL; P = .003, and .0001, respectively). Urinary excretion of 8-isoprostane was slightly
but not significantly decreased in preeclampsia (1200 227 pg/mL), in comparison with urinary excretion
in nonpregnant women (1625 364 pg/mL) and normotensive pregnant women (2149 432 pg/mL). Salivary
concentrations of 8-isoprostane were increased in normotensive women (496 113 pg/mL), in comparison
with nonpregnant women (150 27 pg/mL) but were not related to preeclampsia (419 96 pg/mL;, P .003).
CONCLUSION: Free 8-isoprostane concentrations are increased in the plasma of women with severe preeclampsia. Further studies are warranted to determine whether such increases are caused by an increase
in phospholipase A2 activity, or by lipid peroxidation, or by a decrease in renal excretion. (Am J Obstet Gynecol 2000;183:874-7.)

Key words: Isoprostane, pregnancy, preeclampsia

Preeclampsia is characterized by an increase in maternal systemic vascular resistance, platelet activation, and a
reduction in placental blood flow.1 The primary cause of
this disorder has yet to be elucidated; however, consistent
pathophysiologic findings include generalized maternal
endothelial activation and injury, vasospasm, and microthrombus formation.2 Free radicals derived primarily
from oxygen are increasingly implicated in the pathogenesis of a number of human diseases, including cancer,
atherosclerotic cardiovascular disease, neurodegenerative disorders, and even the normal aging process.3 Preeclampsia is associated with increased activation and generation of oxygen-free radicals by circulating leukocytes.4
A central feature of oxidant injury is peroxidation of
From the Department of Obstetrics and Gynecology, The University of
Tennessee, Memphis.
Presented at the Twentieth Annual Meeting of the Society for MaternalFetal Medicine, Miami Beach, Florida, January 31February 5, 2000.
The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, the Department of Defense, or the United States Government.
Reprint requests: Baha M. Sibai, MD, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, 853 Jefferson Ave, Memphis, TN 38103.
Copyright 2000 by Mosby, Inc.
0002-9378/2000 $12.00 + 0 6/6/108877
doi:10.1067/mob.2000.108877

874

lipids. Oxygen radicals and lipid peroxides are highly reactive compounds that are capable of damaging endothelial cells. A number of studies have found that
plasma levels of lipid peroxidation products5, 6 and the
levels of antibodies to oxidized low-density lipoproteins7
are elevated in preeclampsia compared with levels in normal pregnancy. Although lipid peroxidation appears to
be increased in established preeclampsia, it is not known
whether it is a causative factor or, rather, a manifestation
of the disorder.
Recently, a new group of prostaglandin-like compounds known as isoprostanes have been shown to be
produced in vivo by the action of free oxygen radicals on
arachidonic acid bound to the phospholipid plasma
membrane.8, 9 The isoprostanes are subsequently released by the action of phospholipase A2. In plasma the
isoprostanes circulate in both the free and the lipoprotein-bound state. Free isoprostane is also found in urine.
The most abundant of the isoprostane products of free
radical oxidation in plasma is 8-epi-prostaglandin F2
(8-isoprostane). Quantitation of 8-isoprostane in biologic
fluids and tissues provides an important advance in the
detection and measurement of lipid peroxidation in vivo
and should lead to a better understanding of the mechanisms responsible for oxidant stressrelated alterations in

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homeostasis.10, 11 Our objective was to measure and compare plasma, urinary, and salivary 8-isoprostane concentrations in women with normotensive pregnancies and
the respective concentrations in pregnancies complicated by preeclampsia.
Material and methods
Study population. The following 3 patient groups were
studied: healthy, nonpregnant volunteers (n = 10), normotensive pregnant women (n = 20), and patients with
preeclampsia (n = 40). Patients were recruited from the
outpatient clinics and wards of the Regional Medical Center at Memphis. The study was approved by the University
of Tennessee School of Medicine Institutional Review
Board. After a woman gave informed consent, a blood
sample was collected for measurement of plasma total
and free 8-isoprostane concentrations. A 24-hour urine
sample was collected for measurement of the free 8-isoprostane concentration. Saliva was collected for measurement of the free 8-isoprostane concentration. All pregnant volunteers had intact membranes and a live fetus.
Exclusion criteria for pregnant patients included a diagnosis of chronic hypertension, hepatic or renal dysfunction, asthma, or diabetes. Mild preeclampsia was diagnosed with a systolic blood pressure >140 mm Hg but
<160 mm Hg and with a diastolic blood pressure >90 but
<110 mm Hg, and severe preeclampsia was diagnosed
with a systolic blood pressure 160 mm Hg or a diastolic
blood pressure 110 mm Hg in the presence of proteinuria (protein concentration >300 mg/24 h or >100
mg/dL on a random specimen).
All patients with preeclampsia underwent venipuncture before initiation of seizure prophylaxis with magnesium sulfate. A 10-mL sample of blood was collected in a
tube containing ethylenediaminetetra-acetic acid (1 mg/
mL) and centrifuged immediately at 1500g for 10 minutes.
Plasma was separated and protected from oxidation by
storage at 70C. A 24-hour urine collection was also obtained, and aliquots of a 10-mL sample were stored at
70C. In addition, 1 mL of saliva was collected from each
subject and stored at 70C.
Plasma, urinary, and salivary free 8-isoprostane measurements. To plasma, urine, or saliva (0.5 mL) 5000 cpm
of tritium-labeled prostaglandin F2 was added to enable
estimation of the percentage of recovery. Samples were
then deproteinated with absolute ethanol (2 mL) and
centrifuged at 1500g for 10 minutes. The supernatant was
diluted with 8 mL of deionized water and acidified to
a pH 4.0 with hydrochloride. Each sample was then
passed through a C-18 Supelclean (Supelco, Inc, Bellefonte, Pa) cartridge prewashed with methanol (5 mL)
and deionized water (5 mL). The cartridges were rinsed
with 5 mL deionized water, followed by 5 mL hexane, and
the 8-isoprostane was eluted with 5 mL ethyl acetate containing 1% methanol. The eluate was evaporated to dry-

ness under a stream of dry nitrogen or by means of a centrifugal evaporator. Plasma and saliva samples were reconstituted in 1 mL enzyme immunoassay buffer. Urine
samples were reconstituted with 0.1 mL acetone and further purified by silica gel thin-layer chromatography in
chloroform/methanol/acetic acid/water (80:18:1.0:0.8).
The 8-isoprostane spot was eluted with absolute ethanol,
evaporated to dryness, and then reconstituted in 1 mL
enzyme immunoassay buffer.
Plasma total 8-isoprostane measurements. Lipoproteinbound plasma 8-isoprostane was subjected to alkaline hydrolysis after protein precipitation with absolute ethanol
by adding 1 mL 15% potassium hydroxide to the supernatant and incubating it at 40C for 1 hour. After cooling,
the solution was diluted to 10 mL with deionized water
and adjusted to pH 4.0 with hydrochloride. The 8-isoprostane was then extracted with C-18 cartridges as described earlier in this article for plasma, urinary, and salivary free 8-isoprostane.
Enzyme immunoassay of 8-isoprostane. All samples
were assayed with a commercially available kit (Cayman
Chemical Co, Ann Arbor, Mich). A 50-L sample was assayed in duplicate according to kit instructions. Serial dilutions were assayed when the concentration of 8-isoprostane exceeded the limits of the assay (500 pg/mL).
The antibodies and components used in this enzyme immunoassay were previously validated for the measurement of plasma and urinary 8-isoprostane by means of
negative ion chemical ionization gas chromatography
mass spectrometry.12 The intra-assay and interassay coefficients of variation are 10%. The recovery of 8-isoprostane after extraction averaged 75%.
Data analysis. Unequal variances did not allow the assumption of normality of our data, and therefore 8-isoprostane concentrations were analyzed by 1-way analysis
of variance after transformation to log 10. Post hoc comparisons between pairs of means were made with the use
of the Fisher protected least significant difference test.
A P value < .05 was considered significant.
Results
There were no significant differences in maternal age,
race, or parity among the groups studied. The mean gestational ages at enrollment were as follows: normotensive
group, 23 weeks 1 day (range, 11 weeks33 weeks 5 days);
mild preeclampsia group, 35 weeks 4 days (range, 27
weeks40 weeks 4 days); and severe preeclampsia group,
35 weeks (range, 24 weeks 5 days40 weeks 5 days). Table
I summarizes the 8-isoprostane concentrations in various
fluids among the study groups. Plasma free 8-isoprostane
concentrations were higher in women with severe preeclampsia than in nonpregnant women, normotensive
pregnant women, and women with mild preeclampsia
(P = .003, .001, and .03, respectively). Free 8-isoprostane
concentrations did not differ between nonpregnant

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Am J Obstet Gynecol

Table I. Mean (SEM) concentrations of 8-isoprostanes in plasma (free and total), urine, and saliva
8-Isoprostane concentration (pg/mL)

Nonpregnant
Normotensive pregnancy
Mild preeclampsia
Severe preeclampsia

Plasma free

Plasma total

Urine

Saliva

129 17* (n = 10)

150 11* (n = 20)
174 23* (n = 8)
342 50* (n = 29)

503 70 (n = 9)
472 43 (n = 17)
557 144 (n = 6)
545 139 (n = 28)

1625 364 (n = 9)
2149 432 (n = 19)
1750 684 (n = 8)
1200 227 (n = 19)

150 27 (n = 10)
496 113 (n = 16)
518 123 (n = 6)
419 96 (n = 20)

*P < .05, severe preeclampsia compared with other groups.

P < .05, severe preeclampsia compared with normotensive pregnancy.
P < .005, all comparisons with nonpregnant women.

women and normotensive pregnant women. Plasma total

(free and esterified) 8-isoprostane concentrations did
not differ among the groups studied. Urinary excretion
of 8-isoprostane was decreased in pregnancies complicated by severe preeclampsia, in comparison with normotensive pregnancies (P = .03). Salivary secretion of
8-isoprostane was greater in all pregnancy groups than in
nonpregnant women (P .003).
Comment
To our knowledge only 3 studies to date have investigated plasma or urinary levels of isoprostanes in women
with preeclampsia.5, 13, 14 Our findings of increased free
8-isoprostane levels in the plasma and decreased 8-isoprostane levels in the urine of women with preeclampsia
are consistent with those of other investigators. The 24hour urinary protein concentrations did not seem to correlate with urinary excretion of 8-isoprostanes. However,
in some patients both measurements were not performed, and therefore we cannot comment definitively.
Barder et al14 demonstrated higher levels of plasma free
8-isoprostane levels in women with preeclampsia (72.2
16.3 pg/mL) than in healthy antepartum subjects (33.5
7.2 pg/mL) and a decrease in levels by 6 weeks post partum. Nevertheless, plasma total 8-isoprostane concentrations were not significantly elevated in their patients with
preeclampsia before delivery, but concentrations did fall
significantly post partum (P < .05); those in normal pregnant patients did not change.14 We found the concentrations of free 8-isoprostane in our patients to be much
higher than those reported by Barder et al14 despite the
use of similar immunoassay techniques; results were not
altered when patients with the HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count; n = 2)
were analyzed separately. We do not know why this discrepancy exists, but it could have been caused by differences in either ethnicity or nutritional status of the patient populations or by differences in both of these.
Isoprostanes have been measured in body fluids other
than urine and plasma (ie, synovial fluid and tracheobronchial aspirates). We chose to quantitate salivary 8-isoprostane and to evaluate its utility as a noninvasive, relatively simple indicator of oxidative stress. Although we

were unable to demonstrate a significant difference in secretion of salivary 8-isoprostanes in mild or severe preeclampsia, the overall increase seen in pregnancy is comparable to findings by Wang et al,6 who demonstrated
increased plasma free 8-isoprostanes in normal pregnancies. These findings suggest that even normal pregnancy
may be a state of relative oxidative stress.
8-Isoprostanes have been shown to exert potent vasoconstrictive activity in the lung, kidney, cerebral arterioles, and hepatic portal vein, apparently acting through
thromboxane A2endoperoxide (prostaglandin H2) receptors.15 This finding could explain both the decrease
in renal clearance of free 8-isoprostane seen in preeclampsia and the increase in plasma levels. Secretion of
endothelin 1 is increased in preeclampsia13 and stimulated by isoprostanes.16 Preliminary work by Fukunaga
et al16 and also by Yura et al17 suggests that a novel isoprostane receptor exists. Molecular identification and
cloning of this putative receptor will add greatly to the
study of the mechanism(s) by which isoprostanes influence endothelial activity.
The question remains as to whether plasma levels of
free 8-isoprostane contribute directly to the pathogenesis
of preeclampsia.
Our results and those of other investigators suggest
that preeclampsia is associated with increased plasma
concentrations of free 8-isoprostane and increased lipid
peroxidation. The high levels of free 8-isoprostane seen
in preeclampsia, however, may be the result of widespread oxidant vascular damage occurring in established
severe disease, or they may be involved in the pathogenesis of the condition and predate its clinical expression. To
date, no studies have investigated the plasma levels of
8-isoprostane or phospholipase A2 activity in patients with
preeclampsia before clinical diagnosis. Longitudinal
studies of 8-isoprostane levels in normal pregnancy and
in preeclampsia will address this important issue.
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