J. Obstet. Gynaecol. Res. Vol. 28, No. 5: 274–279, October 2002

274

Blackwell Science, LtdOxford, UKJOGThe Journal of Obstetrics and Gynaecology Research1341-80762002 Asia and Oceania Federation of Obstetrics and Gynaecology

285October 2002

040Anti-oxidants in former pre-eclamptic patientsH. Ozan, Y. Ilçöl, Y. Kimya, C. Cengiz, B. Ediz

10.1046/j.1341-8076.2002.00040.x

Original Article274279BEES SGML

Received: 10 April 2001. Accepted: 5 July 2002.Reprint requests to: Associate Professor Hakan Ozan, Ibrahimpasa Mah, Inanç Sok, Özpirinç Apt, A Blok, No: 20/5, 16010 Bursa, Turkey. Email: hozan@superonline.com

Plasma anti-oxidant status and lipid profile in non-gravida women with a history of pre-eclampsia

Hakan Ozan

1

, Yesim .Ilçöl

2

, Yalçin Kimya

1

, Candan Cengiz

1

and Bülent Ediz

3

1

Department of Obstetrics and Gynaecology,

2

Department of Biochemistry and

3

Department of Statistics, Medical Faculty, Uludag University, Bursa, Turkey

Abstract

Objective:

To investigate the total plasma anti-oxidant status, the plasma lipid profile and the uterine arteryDoppler velocity waveform in formerly pre-eclamptic women.

Methods:

Thirty-two formerly pre-eclamptic, non-gravida women constituted the study group, while 28 age-matched non-gravida women who had never had pre-eclampsia served as control subjects. On days 17–19 oftheir menstrual cycle, fasting plasma samples were collected for total anti-oxidant status (TAS) and lipid pro-file evaluation, and uterine artery Doppler velocity waveform studies were performed. Results were analyzedwith Mann–Whitney

U

-test and Pearson correlation analysis.

Results:

There was no significant difference between the groups in means of the uterine artery Doppler veloc-ity waveforms and the plasma lipid levels, but body mass index values were significant (

P

<

0.005). The TASvalue was subnormal in 72% of the formerly pre-eclamptic group and in 35% of the control group. The meanplasma TAS value was 1.20

±

0.05 mmol/L and was significantly lower in the study group when comparedwith the control group (

P

<

0.05).

Conclusion:

The current study reveals significantly decreased TAS in women with a history of pre-eclampsia,which may have an important role in pathophysiology.

Key words:

antioxidant, Doppler, lipid, pre-eclampsia.

Introduction

Pre-eclampsia is an important cause of fetomaternalmorbidity and mortality. It is not only unpredictable inonset and progression, but also has no effective man-agement strategy except termination of pregnancy.

1

An increase in lipid pre-oxidation products and adecrease in anti-oxidant activity have been reported inpre-eclampsia.

2

Lipid peroxides formed at a primarysite are then transported through the circulation bylipoproteins and cause damage at distant tissues tocause endothelial dysfunction and hence an increasein sensitivity to vasopressors.

3,4

Researchers of pre-

eclamptic women have also shown that the lipid per-oxides induce a long-lasting activation of leukocytesand in turn cause the generation of superoxide anionswhich inactivate endothelium-derived relaxing factorand contribute to vasoconstriction.

5,6

However, thereports about anti-oxidant levels in pre-eclampsia areconflicting.

7

Whatever the concentration of oxidantswhen compared with that of the normal pregnancy is,there would be absolute or relative deficiency of anti-oxidants that would explain the development ofpre-eclampsia.

On the other hand, women with pregnancies com-plicated by pre-eclampsia, as compared with normo-

Anti-oxidants in former pre-eclamptic patients

275

tensive pregnant women, experience more pronoun-ced elevations in free fatty acids, triglycerides, and lowdensity lipoprotein (LDL) cholesterol, with a fall inhigh density lipoprotein (HDL) cholesterol levels.

8,9

The state of hypertriglyceridemia and HDL cholesteroldeficiency may be associated with hypercoagulabilitydue to activation of coagulation factors, increasedplatelet aggregation, increased platelet collagen adhe-sion and activation of plasminogen activator inhibitor-1 and constriction of placental vasculature due toreduced prostacyclin synthesis.

10–13

In uncomplicated pregnancies, trophoblastic inva-sion of maternal spiral arteries causes an increase inuterine blood flow and a fall in resistance that iscomplete by about 24 weeks of gestation.

14

In pre-eclampsia, because the trophoblastic invasion is defec-tive, the uteroplacental circulation remains in a state ofhigh resistance.

15

Hence both oxidant–anti-oxidantimbalance due to lipid peroxide formation and per-fusion disruption due to lipid profile changes and pla-cental implantation defects in pregnancy can makewomen prone to pre-eclampsia.

Therefore, studies based on possible etiologicalfactors have been focused on the development of pre-dictive tests for pre-eclampsia.

16

Early discriminationof high-risk and low-risk women for pre-eclampsiawould prevent unnecessary intensive antenatal careand would allow early therapeutic intervention to pre-vent progression when necessary. Though many differ-ent screening tests have been proposed for prediction,they are either not sensitive enough or are sensitivelate in pregnancy.

17

However, a great majority of thosestudies have been done during pregnancy, thereforethey have been prone to the physiological changes ofthe pregnancy. Because an ideal screening test wouldlead to the recognition of high-risk women beforepregnancy, in this controlled study, we investigated theplasma total anti-oxidant status (TAS), the lipid profileand the uterine artery Doppler velocity waveforms inwomen who had a history of pre-eclamptic pregnancy.

Materials and Methods

After the evaluation of medical records, 187 womenwho had been admitted to the Obstetrics Departmentof Uludag University Medical Faculty, Bursa, Turkeywith signs and symptoms of severe pre-eclampsiabetween 1 January 1995 and 31 December 1999 werecalled back to participate in the study. The diagnosisof pre-eclampsia in those patients had been establishedin accordance with the definitions of the American Col-

lege of Obstetricians and Gynecologists.

18

Thirty-twoof those women who were non-gravida and had nogestation following the last admission to our hospitalconstituted the study group, while 28 age-matched,non-gravida women, who never had pre-eclampsia,served as control subjects. All participants had normalmenstrual cycles and none of them had any medicalproblems or were taking any medication or vitaminsupplementation. The aim and the methodology ofthe study was explained and informed consent wasreceived from all participants.

The women were invited to participate in the studyon days 17–19 of their menstrual cycle. After 10 hoursof fasting, venous blood samples were drawn inheparin-coated 10 mL vacutainer tubes and wereimmediately centrifuged at 3000 r.p.m. for 5 min atroom temperature to separate plasma. The plasma wasaliquoted into three 1 mL Ependorf tubes, wrapped inaluminum foil to protect against photo-oxidation, andthe samples were stored at

-

80

°

C until analysis, within3 months of sampling.

Plasma samples were assayed for total anti-oxidantstatus (Randox Total Anti-oxidant Status Kit, RandoxLaboratories, United Kingdom). The assay is based onthe incubation of 2,2’-azino-di-(3-ethylbenzthiazolinesulfonate) with a peroxidase (metmyoglobin) andH

2

O

2

to produce the radical cation ABTS

+

which has arelatively stable blue-green color, measured at 600 nm.Anti-oxidants in the added sample cause suppressionof this color production to a degree, which is propor-tional to their concentration. The suppression of thecolor is compared to that of the standard provided. Thelinear limit of the method is 2.5 mmol/L and its sensi-tivity is 0.01 mmol/L. The plasma reference range forEuropean population is 1.30–1.77 mmol/L.

Plasma total cholesterol (TC) and triglyceride(TG) levels were measured by automated enzymaticmethods using cholesterol oxidase, glycerol kinaseand glycerophosphate oxidase (Biotrol reagent kit,Biotrol Diagnostics, France). Plasma HDL cholesterollevel was determined by immunoinhibition method(Randox reagent kit, Randox Laboratories, UnitedKingdom). Low density lipoprotein cholesterol andvery low density lipoprotein (VLDL) cholesterolconcentrations were derived using the Freidewaldequation.

19

Blood pressure was taken after 30 min rest in asitting position from the right arm with a standardmercury sphygmomanometer using phases 1 and 4of the Korotkoff sounds for the systolic and diastolicblood pressures, respectively. Toshiba SSH 140 A,

H. Ozan

et al.

276

color-pulsed Doppler with 5 MHz transvaginal probeand ultrasonographic intensity less than 100 mW/cm

2

was used for uterine artery velocimetry. The mainuterine artery was located at the uterocervical junctionon both sides. Flow velocity waveforms were re-corded to measure the pulsatility index (PI

=

systolicvelocity – diastolic velocity/mean velocity), the resis-tance index (RI

=

systolic velocity – diastolic velocity/systolic velocity), and the systolic

−

diastolic ratio (S/D

=

systolic velocity/diastolic velocity).Gestational age at delivery, date of the last delivery,

body mass index (BMI), birth weight of the newborn,5th minute Apgar score, arterial blood pressure, hema-tocrit, white blood cell count, thrombocyte count,blood urea nitrogen, serum uric acid, serum creatinine,serum albumin, serum glutamic oxaloacetic transami-nase and serum glutamic pyruvic transaminase levelsduring admission to our hospital with the diagnosisof pre-eclampsia were retrieved from the medicalrecords.

The plasma total anti-oxidant status level, theplasma TC, HDL cholesterol, LDL cholesterol, VLDLcholesterol and TG levels and the uterine artery PI, RIand S–D-values of the groups were compared usingMann–Whitney

U

-test and any correlation betweenthose parameters was evaluated with Pearson correla-tion analysis. The results were given as mean

±

SE andthe level of significance was determined as

P

<

0.05.

Results

Characteristics of the patients are shown in Table 1.The mean age, the mean gravida, the mean para andthe mean abortion values did not differ significantlybetween the groups (NS). In the formerly pre-eclamptic group the mean gestational age at deliverywas 242.6

±

3.8 days, the mean birth weight was2170.0

±

111.4 g, and the mean 5th minute Apgar scorewas 5.5

±

0.3. The mean time period between the deliv-ery and the date of study was 40.2

±

2.9 and 40.9

±

2.6months in the study and the control groups, respec-tively (NS).

The mean arterial systolic but not the diastolic bloodpressure was significantly higher in the study groupwhen compared with the control group (

P

<

0.05) (seeTable 2). In the study group, both the mean arterial sys-tolic and diastolic blood pressures that had beenrecorded during pre-eclampsia were higher than thosemeasured at the time of the study (

P

<

0.05) (seeTable 2). The mean PI, the mean RI and the mean S/Dratio measurements differed significantly neitherbetween the groups nor between the right and leftuterine arteries within the groups (NS) (see Table 2).

There was no difference between the mean plasmaTC, the mean plasma HDL cholesterol, the meanplasma LDL cholesterol, the mean plasma VLDL cho-lesterol and the mean plasma TG levels (NS) (seeTable 3). The mean BMI was significantly higher in theformerly pre-eclamptic group (

P

<

0.005). However, theplasma lipids did not show any significant correlationwith the BMI and the Doppler velocity waveformmeasurements (NS).

The

mean

plasma

TAS

value

was

1.20

±

0.05 mmol/Lin the study group and was within the normal rangein the control group; the difference in between was sta-tistically significant (

P

<

0.05) (see Table 3). The plasmaTAS value was not correlated with age, gravida, para,arterial blood pressure, plasma HDL, VLDL and TGlevels and the Doppler velocity waveform measure-ments in both groups (NS). However, it was signifi-cantly correlated with plasma LDL (

r

=

−

0.446,

P

<

0.05)and TC (

r

=

−

0.456,

P

<

0.05) levels and the gestationalage at delivery (

r

=

0.469,

P

<

0.05) in the formerly pre-eclamptic group. When the cut-off value was acceptedas 1.30 mmol/L, the sensitivity was 0.6970, the speci-ficity was 0.6670, the positive predictive value was0.7188 and the negative predictive value was 0.6429.

In the formerly pre-eclamptic group the mean hema-tocrit was 35.0

±

0.7%, the mean white blood cell countwas 13 065.4

±

677.7/mL, the mean thrombocyte countwas 13 7115.4

±

12 808.2/mL, the mean blood ureanitrogen was 39.1

±

3.5 mg/dL, the mean serum uricacid was 7.5

±

0.4 mg/dL, the mean serum creatininewas 0.99

±

0.05 mg/dL, the mean serum glutamic

Table 1

The age and the gestational characteristics of the groups (mean

±

SE)

Age*

(years) Gravida* Para* Abortion* BMI**

(kg/m

2

) Duration

d-s

* (months)

Study group (

n

=

32) 32.7

±

1.3 2.3

±

0.3 1.8

±

0.2 0.6

±

0.1 24.4

±

0.2 40.2

±

2.9Control group (

n

=

28) 33.1

±

1.1 2.2

±

0.2 1.4

±

0.2 0.8

±

0.1 23.2

±

0.3 40.9

±

2.6

BMI, body mass index; Duration

d-s

, duration between the last delivery and the study.*P

>

0.05; **P

<

0.005.

Anti-oxidants in former pre-eclamptic patients

277

oxaloacetic transaminase was 102.4

±

28.1 U/L, themean serum glutamic pyruvic transaminase was62.0

±

16.3 U/L, the mean serum albumin was2.7

±

0.1 g/dL during admission with the diagnosis ofpre-eclampsia and there was no correlation betweenthose parameters and the plasma TAS value, the lipidprofile, BMI and the Doppler velocity waveformmeasurements (NS).

Discussion

Contrary to the great majority of studies, there aresome conflicting reports about anti-oxidant levels inpre-eclampsia. Morris

et al

. have reported the circu-lating levels of oxidative stress markers and lipidperoxides to be elevated both in pre-eclampsia anduncomplicated pregnancies with no significant dif-ference in between.

7

Whatever the concentration ofoxidants when compared with that of the normal preg-nancy, there would be absolute or relative deficiencyof anti-oxidants that would explain the developmentof pre-eclampsia. Though there are many studies indi-cating this deficiency in the literature, they are usuallyfocused on a certain part of the anti-oxidative systemsuch as vitamin E, vitamin C,

β

-carotene, malondialde-hyde, glutathione peroxidase and super-oxide dismu-tase and therefore report conflicting results.

2,4,20,21

Allthose anti-oxidants work in collaboration in the humanbody and deficiency of one of them does not necessi-tate the deficiency of the others, and indeed may causecompensatory increments in the activity of the remain-ing anti-oxidants. Measurement of total anti-oxidantstatus, on the other hand, can provide information onan individual’s overall anti-oxidant capacity, whichmay include those anti-oxidants not yet recognized ornot easily measured. In our study, though we were notable to show any correlation with the blood pressuresof the former pre-eclamptics, the mean TAS value ofthe study group was significantly higher than that ofthe control group. This difference may be due togenetic, nutritional or environmental factors thatwould be disclosed by the forthcoming studies. Onthe other hand, the significant negative correlationsbetween the plasma TAS value and the plasma LDLand TC levels may be due to consumption of plasmaanti-oxidants by lipid peroxides that were triggeredby LDL and TC molecules in former pre-eclampticsand make them prone to pre-eclampsia.

Women with pregnancies complicated by pre-eclampsia, as compared with normotensive pregnantwomen, experience more pronounced elevations in

Tab

le2

The

art

eria

l blo

od p

ress

ures

and

the

ute

rine

art

ery

Dop

pler

vel

ocit

y w

avef

orm

mea

sure

men

ts o

f th

e gr

oups

(m

ean

±

SE

)

Blo

od p

ress

ure

AD

(mm

Hg)

Blo

od p

ress

ure

AS

(mm

Hg)

R

ight

ute

rine

art

ery

Lef

t ut

erin

e ar

tery

Syst

olic

Dia

stol

ic

Syst

olic

**

Dia

stol

ic*

PI*

RI*

S/D

*PI

*R

I*S/

D*

Stud

y gr

oup

(

n=

32)

175.

6±

3.8

110.

6±

2.5

121.

9±

2.7

76.6

±1.

81.

84±

0.09

0.72

±0.

013.

73±

0.14

1.86

± 0.

080.

73±

0.01

3.74

±0.

16

Con

trol

gro

up(n

=28

)–

–11

2.9

±1.

772

.5±

1.7

1.80

±0.

080.

71±

0.01

3.46

±0.

111.

87±

0.07

0.73

± 0.

013.

56±

0.13

Blo

od p

ress

ure A

D, b

lood

pre

ssur

e at

del

iver

y; B

lood

pre

ssur

e AS,

blo

od p

ress

ure

at s

tud

y; P

I, pu

lsat

ility

ind

ex; R

I, re

sist

ance

ind

ex; S

/D

, sys

tolic

–dia

stol

ic r

atio

.*P

>0.

05; *

*P<

0.05

.

H. Ozan et al.

278

free fatty acids, triglycerides, and LDL cholesterol,with a fall in HDL cholesterol levels.8,9 Though thereis almost complete consensus on lipid profile in pre-eclampsia, the question is if this change in lipid profileof pre-eclamptic women exists before pregnancy ordevelops during pregnancy to contribute to the risk offuture pre-eclampsia. Lorentzen et al. have shown anelevation in free fatty acids and triglycerides at 18weeks of gestation in women who later develop pre-eclampsia.10 In a controlled study in 1999, He et al.reported higher levels of systolic and diastolic bloodpressures in non-pregnant women with a historyof pre-eclampsia.22 Though in the follicular phaseplasma levels of all lipids and lipoproteins were simi-lar in both groups, the luteal phase total cholesterol,triglycerides and VLDL cholesterol were found to beelevated significantly in the formerly pre-eclampticwomen. Because the luteal phase is important forimplantation and trophoblast invasion, and is associ-ated with a proposed increase in plasma lipid levels,we took the plasma samples in the luteal phase. How-ever, our results did not confirm He et al. and showedno difference in means of plasma lipid levels betweenthe study and the control groups. The mean BMIwas significantly higher in the formerly pre-eclampticgroup; it is reported to be a validated risk factor forpre-eclampsia independent of baseline systolic anddiastolic blood pressure measurements.23,24 The sig-nificantly higher mean arterial systolic blood pressurein the study group was considered to be incidentalbecause it was not correlated significantly with anyother parameters that were the subjects of the studyand there were no significant difference between themean arterial diastolic blood pressures.

Harrington et al., using pulsed wave Doppler ultra-sound to evaluate the resistance of the uterine artery,found uterine artery resistance increased in pre-eclampsia.25 However, there has been a disagreementon where to look for uterine artery waveforms: unilat-erally or bilaterally on the placental or on the non-placental side of the uterus. Differences in gestational

age at the time of examination may also affect theresults. If the defect in trophoblastic invasion in pre-eclampsia is hypothesized to be due to uterine factorssuch as local oxidant–anti-oxidant imbalance, increasein lipid concentration and development of atherosis inspiral arteries, uterine artery Doppler velocimetry,without the effects of placenta and gestational age,would give useful data for the prediction of pre-eclampsia in non-pregnant women. In our study, themean PI, the mean RI and the mean S/D values of bothright and left uterine arteries did not show any signif-icant difference between the study and the controlgroups.

The current study reveals decreased total anti-oxidant capacity in women with a history of severepre-eclampsia. This fact may be due to many factorssuch as malnutrition, poor socioeconomic condition orinborn errors of metabolism. Whatever the cause is, itwould lead to insufficient neutralization of oxidantsand would make the women prone to the developmentof pre-eclampsia. Inability to find any differencebetween the uterine artery flow velocity waveformsmay be associated with indifferent plasma lipid levelsand probably similar tendencies for atherosis in bothgroups. Our results may provide some implications forthis subject, but further prospective studies withlarge numbers of women are needed to decide if theplasma TAS is already decreased in women who laterbecome pregnant and develop pre-eclampsia.

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Table 3 The plasma lipid profile and the total anti-oxidant status values of the groups (mean ± SE)

TC*(mg/dL)

HDL*(mg/dL)

LDL*(mg/dL)

VLDL*(mg/dL)

TG*(mg/dL)

TAS**(mmol/L)

Study group (n = 32) 148.8 ± 7.0 33.3 ± 0.8 95.9 ± 6.9 19.6 ± 2.2 98.0 ± 10.8 1.20 ± 0.05Control group (n = 28) 147.4 ± 6.6 31.3 ± 0.8 96.1 ± 5.6 20.4 ± 2.3 101.2 ± 11.7 1.40 ± 0.05

TC, total cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very-low density lipoprotein; TG, triglyceride; TAS, total anti-oxidant status.*P > 0.05; **P < 0.05.

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279

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