7/30/2019 smw-10397

1/5

Increased oxidative stress in patientswith hydatidiform mole

Muge Harmaa, Mehmet Harmaa, Ozcan Erelb

a Departments of Gynaecology and Obstetrics, University of Harran, Faculty of Medicine,Sanliurfa, Turkey

b Biochemistry Department, University of Harran, Faculty of Medicine, Sanliurfa, Turkey

Reactive oxygen species (ROS) are producedin metabolic and physiological processes, andharmful oxidative reactions may occur in organ-isms which remove ROS via enzymatic and non-

enzymatic antioxidative mechanisms. Under someconditions increases in oxidants and decreases inantioxidants cannot be prevented, and the oxida-tive/antioxidative balance shifts towards the oxida-tive status. Oxidative stress, which has been impli-cated in over 100 disorders, develops in conse-quence [1].

Blood contains many antioxidant moleculesthat prevent and/or inhibit harmful free radical re-actions [2]. Plasma concentrations of antioxidantscan be measured separately in the laboratory, butthese measurements are time-consuming, labour-

intensive and costly. Since antioxidative effectsof antioxidant components of plasma are additive,the measurement of total antioxidant potential(TAOP) reflects the antioxidative status of plasma.

We evaluated the total antioxidative status ofplasma with TAOP [3, 4].

Hydrogen peroxide and other derivatives ofperoxides, produced physiologically in organismsand occurring in higher concentrations in somepathological conditions, diffuse into plasma. Here,

antioxidant components of plasma overwhelmthem and they are consumed [5]. We evaluated thetotal oxidative status of plasma by measuring totalperoxide level [6].

It is known that oxidative stress increases dur-ing normal pregnancy. In healthy pregnancy it hasbeen reported that plasma lipid hydroperoxide lev-els are increased and total antioxidant capacity de-creased [7], while erythrocyte glutathione peroxi-dase activity and its cofactor selenium are dimin-ished [8]. However, the nature of this mechanismis not yet known.

Pre-eclamptic patients are exposed to in-creased oxidative stress [9, 10], as are patients withcomplicated pregnancies such as those involvinghypertension and diabetes mellitus [11, 12]. Pla-cental hypersecretion of lipid peroxides or de-creased placental antioxidant enzyme production

Objective:The aim of this study was to deter-mine the oxidative status and antioxidative status

of plasma of patients with complete hydatidiformmole (CHM) and to compare these values withnormal pregnancy.

Method:Thirty-eight patients with CHM and31 healthy pregnant women were enrolled in thestudy. To determine the antioxidative status ofplasma, total antioxidant potential (TAOP) wascalculated, and to determine the oxidative status ofplasma total peroxide levels were measured. Theratio of TAOP to total peroxide was accepted as anindicator of oxidative stress.

Results: TAOP of plasma was significantly

lower in patients with hydatidiform mole than inhealthy pregnant women [mean (SD) values were511.9 (105.8) and 571.7 (109.4) mol Troloxequiv./L respectively (p

7/30/2019 smw-10397

2/5

may lead to endothelial dysfunction. Insufficientantioxidant capacity may lead to excess oxidativestress; oxidative injury may subsequently occur inmaternal and placental compartments [13]. Thus,in pre-eclampsia, placental abnormality and asso-ciated metabolic changes cause increased oxidativestress [14]. Similar metabolic changes are presentin molar pregnancy.

We postulated that increased oxidative stressmay be present in patients with CHM, and in thepresent state of knowledge there has been no re-port involving the oxidative/antioxidative status ofplasma in the pseudopregnant state of CHM. Thisstudy is an attempt to add to that body of knowl-edge.

Oxidative stress in hydatidiform mole 564

Materials and methods

Subjects

This study involved 69 women who attended HarranUniversity Hospital during the period between July 1998and September 2002. Of these, 31 were healthy pregnantwomen (controls) in the first trimester of pregnancy witha single viable foetus (mean gestational age 13.2 weeks asestimated by ultrasonography). The remaining 38 patientshad CHM (mean gestational age 12.9 weeks as estimated

by last menstrual period). Obese women, underweightwomen and smokers were excluded from the study. Diag-nosis of complete hydatidiform mole was based onhistopathological examination of molar tissue, showingcharacteristically abnormal proliferation of trophoblastictissue, lack of an identifiable foetus, chorionic villi withgeneralised hydatidiform swelling, and diffuse tropho-blastic hyperplasia resulting from abnormal fertilisation.Informed written consent was obtained from all subjects.

Samples

Blood samples were withdrawn into heparinisedtubes after overnight fasting. Plasma was separated fromcells by centrifugation at 1500 g for 10 min. Plasma sam-

ples were assayed immediately or stored at 80 C.

Measurement of plasma total antioxidant potential

TAOP of plasma was determined using the ferric re-ducing/antioxidant power (FRAP) assay, developed byBenzie and Strain [3, 4]. Briefly, 1.5 ml of working pre-warmed 37 C FRAP reagent (10 vols 300 mM acetatebuffer, pH 3.6 + 1 vol. 10 mM 2,4,6-tripyridyl-S-triazinein 40 mM HCl + 1 vol. 20 mMol FeCl3) was vortex mixedwith 50 mL test sample and standards. The test was per-formed at 37 C. Absorbance at 593 nm was read againsta reagent blank at a predetermined time (04 min) aftersample-reagent mixing. An arbitrary unit, the Troloxequivalent concentration, was used as the measurement

of TAOP [15]. Thus the results were expressed as mmolTrolox equiv./L [3, 4, 7].

Measurement of plasma total peroxideconcentration

Total peroxide concentrations were determined usingthe FOX2 method [6] with minor modifications. TheFOX2 test system is based on oxidation of ferrous ion toferric ion by various types of peroxides contained withinthe plasma samples, to produce a coloured ferricxylenolorange complex whose absorbance can be measured. The

FOX2 reagent was prepared by dissolving ammonium fer-rous sulphate (9.8 mg) in 250 mM H2SO4 (10 ml) to givea final concentration of 250 mM ferrous ion in acid. Thissolution was then added to 90 ml HPLC-grade methanolcontaining 79.2 mg butylated hydroxytoluene (BHT). Fi-nally, 7.6 mg xylenol orange was added with stirring tomake the final working reagent (250 mM ammonium fer-rous sulphate, 100 mM xylenol orange, 25 mM H2SO4, and4 mM BHT in 90% vol/vol methanol in a final volume of100 ml). The blank working reagent contained only fer-rous sulphate.

Aliquots (200mL) of plasma were mixed with 1800 mLFOX2 reagent. After incubation at room temperature for30 min, the vials were centrifuged at 12000 g for 10 min.

Absorbance of the supernatant was then determined at 560nm. Total peroxide content of plasma samples was deter-mined as a function of the absorbance difference betweentest and blank tubes using a solution of H2O2 as standard.The coefficient of variation for individual plasma sampleswas less than 5%.

Oxidative stress index

The ratio of total peroxide to total antioxidant po-tential was the oxidative stress index, an indicator of thedegree of oxidative stress.

Statistical analysis

Students t test was performed using SPSS package.

P 0.05 was considered statistically significant.

Results

Demographic and clinical data of the subjectsare shown in table 1. There were no differences inmean age, gestational age, gravidity, parity, abor-tion and body mass index (BMI) between patients

with CHM and controls.

As seen in table 2, plasma TAOP levels of pa-tients with CHM were found to be significantlylower than those of healthy pregnant women:

511.9 (105.8) vs 571.7 (109.4) mol Troloxequiv./L (p

7/30/2019 smw-10397

3/5

In the present study, we found that the oxida-tive/antioxidative balance shifted towards oxida-tive status, namely increased oxidative stress waspresent in patients with CHM compared withhealthy pregnant control subjects.

Further studies are needed to explain the exactmechanisms of oxidative stress in patients with

CHM. We postulated that the hypothetical mech-anisms in pre-eclampsia may also be responsiblefor CHM. In both diseases the placenta is a keysource of factors which lead to similar metabolicchanges [10].

In pre-eclampsia leucocytes, such as neu-trophils and monocytes, are activated. Inflamma-tory cytokines, such as interleukin-6 (IL-6) and tu-mour necrosis factor (TNF(alpha)), and the vascu-lar cell adhesion molecule (VCAM-1), are elevatedin the maternal circulation. Activated neutrophilsattach to endothelial cells where they generate

ROS, resulting in oxidative stress within the celland internal milieu [14]. In CHM disease, similar

inflammatory mechanisms to those mentionedabove are present [16, 17]. Increased ROS and de-creased antioxidative defence systems may alsolead to oxidative injury in patients with CHM.

As is seen in table 2, we found that TAOP wasdecreased and the total peroxide level increased inthese patients and, as expected, they are exposed to

increased oxidative stress. Increased oxidativestress may play a role in the pathogenesis of thedisease or may be secondary to the disease. Sup-plementation with antioxidant vitamins C and Emay prove useful in the treatment of CHM.

Correspondence:Muge Harma6. Sokak, 2/9,Bahcelievler, 06500

Ankara,

TurkeyE-Mail: harmam@ixir.com

S W I S S M E D W K LY 2 0 0 3 ; 13 3 : 5 63 5 6 6 w w w . s m w. c h 565

Variables Complete hydatidiform mole Healthy pregnant women P(n = 38) (n = 31)

Mean (SD) Mean (SD)

Age (years) 31.0 (8.1) 29.5 (5.7) NS

Gestational age (weeks) 12.9 (4.8) 13.2 (4.4) NS

Gravidity 5.9 (3.5) 5.1 (2.3) NS

Parity 4.5 (3.3) 3.3 (2.0) NS

Abortion 0.3 (0.6) 0.2 (0.5) NSBMI (kg/m2) 21.1 (1.8) 21.0 (1.7) NS

Complete hydatidiform mole Healthy pregnant women P(n = 38) (n = 31)

Mean (SD) Mean (SD)

Total antioxidant potential 511.9 (105.8) 571.7 (109.4) 0.029(mol Trolox equiv./L)

Total peroxide 21.8 (6.4) 15.6 (6.4) 0.001(mol H2O2/L)

Oxidative stress index 4.43 (1.70) 2.92 (1.50) 0.001

Table 1

Demographic charac-

teristics of patients

with complete hyda-

tidiform mole and

healthy pregnant

women.

Table 2

Plasma indicators of

oxidative stress in

patients with com-

plete hydatidiform

mole (CHM) and

healthy pregnant

controls.

Discussion

References

1 Halliwell B. Free Radicals In Biology And Medicine. Oxford:Oxford Science Publications, 2000.

2 Young IS, Woodside JV. Antioxidants in health and disease. JClin Pathol 2001;54:17686.

3 Benzie IF, Strain JJ. The ferric reducing ability of plasma(FRAP) as a measure of antioxidant power: the FRAP assay.

Anal Biochem 1996;239:706.

4 Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay:direct measure of total antioxidant activity of biological fluidsand modified version for simultaneous measurement of totalantioxidant power and ascorbic acid concentration. MethodsEnzymol 1999;299:1527.

5 Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V.Method for the measurement of antioxidant activity in humanfluids. J Clin Pathol 2001;54:35661.

6 Miyazawa T. Determination of phospholipid hydroperoxides inhuman blood plasma by a chemiluminescence-HPLC assay.Free Radic Biol Med 1989;7:20917.

7 Toescu V, Nuttall SL, Martin U, Kendall MJ, Dunne F. Oxida-tive stress and normal pregnancy. Clin Endocrinol (Oxf) 2002;57:60913.

8 Nakai A, Oya A, Kobe H, Asakura H, Yokota A, Koshino T, et

al. Changes in maternal lipid peroxidation levels and antioxi-dant enzymatic activities before and after delivery. J NipponMed Sch 2000;67:4349.

9 Many A, Hubel CA, Fisher SJ, Roberts JM, Zhou Y. Invasivecytotrophoblasts manifest evidence of oxidative stress inpreeclampsia. Am J Pathol 2000;156:32131.

10 Hubel CA. Oxidative stress in the pathogenesis of preeclamp-sia. Proc Soc Exp Biol Med 1999;222:22235.

7/30/2019 smw-10397

4/5

11 Bowen RS, Moodley J, Dutton MF, Theron AJ. Oxidative stressin pre-eclampsia. Acta Obstet Gynecol Scand 2001;80:71925.

12 Orhan H, Onderoglu L, Yucel A, Sahin G. Circulating bio-markers of oxidative stress in complicated pregnancies. ArchGynecol Obstet 2003;267:18995.

13 Maseki M, Nishigaki I, Hagihara M, Tomoda Y, Yagi K. Lipidperoxide levels and lipids content of serum lipoprotein fractionsof pregnant subjects with or without pre-eclampsia. Clin Chim

Acta 1981;115:15561.14 Merabishvili N, Sanikidze T, Sioridze E. Contemporary Un-

derstanding of Etiopathogenesis of Preeclampsia. Tbilisi StateMedical University Annals of Biomedical Research and Educa-tion 2002;2:95102.

15 Cao G, Prior RL. Comparison of different analytical methodsfor assessing total antioxidant capacity of human serum. ClinChem 1998;44:130915.

16 Shaarawy M, Darwish NA. Serum cytokines in gestational tro-phoblastic diseases. Acta Oncol 1995;34:17782.

17 Prabha B, Molykutty J, Swapna A, Rajalekshmi TN, Ganga-dharan VP. Increased expression of interleukin-1 beta is asso-ciated with persistence of the disease and invasion in completehydatidiform moles (CHM). Eur J Gynaecol Oncol 2001;22:506.

Oxidative stress in hydatidiform mole 566

7/30/2019 smw-10397

5/5

What Swiss Medical Weekly has to offer:

SMWs impact factor has been steadilyrising, to the current 1.537

Open access to the publication viathe Internet, therefore wide audienceand impact

Rapid listing in Medline LinkOut-button from PubMedwith link to the full textwebsite http://www.smw.ch (direct linkfrom each SMW record in PubMed)

No-nonsense submission you submita single copy of your manuscript bye-mail attachment

Peer review based on a broad spectrumof international academic referees

Assistance of our professional statisticianfor every article with statistical analyses

Fast peer review, by e-mail exchange withthe referees

Prompt decisions based on weekly confer-ences of the Editorial Board

Prompt notification on the status of yourmanuscript by e-mail

Professional English copy editing No page charges and attractive colour

offprints at no extra cost

Editorial BoardProf. Jean-Michel Dayer, GenevaProf. Peter Gehr, BerneProf. Andr P. Perruchoud, BaselProf. Andreas Schaffner, Zurich

(Editor in chief)Prof. Werner Straub, Berne

Prof. Ludwig von Segesser, Lausanne

International Advisory CommitteeProf. K. E. Juhani Airaksinen, Turku, FinlandProf. Anthony Bayes de Luna, Barcelona, SpainProf. Hubert E. Blum, Freiburg, GermanyProf. Walter E. Haefeli, Heidelberg, GermanyProf. Nino Kuenzli, Los Angeles, USAProf. Ren Lutter, Amsterdam,

The NetherlandsProf. Claude Martin, Marseille, FranceProf. Josef Patsch, Innsbruck, AustriaProf. Luigi Tavazzi, Pavia, Italy

We evaluate manuscripts of broad clinicalinterest from all specialities, including experi-mental medicine and clinical investigation.

We look forward to receiving your paper!

Guidelines for authors:http://www.smw.ch/set_authors.html

All manuscripts should be sent in electronic form, to:

EMH Swiss Medical Publishers Ltd.SMW Editorial SecretariatFarnsburgerstrasse 8CH-4132 Muttenz

Manuscripts: submission@smw.ch

Letters to the editor: letters@smw.chEditorial Board: red@smw.chInternet: http://www.smw.ch

Swiss Medical Weekly: Call for papers

SwissMedical Weekly

The many reasons why you shouldchoose SMW to publish your research

Official journal of

the Swiss Society of Infectious disease

the Swiss Society of Internal Medicine

the Swiss Respiratory Society

Impact factor Swiss Medical Weekly

0 . 7 7 0

1 . 5 3 7

1 . 1 6 2

0

0. 2

0. 4

0. 6

0. 8

1

1. 2

1. 4

1. 6

1. 8

2

1

9

9

5

1

9

9

6

1

9

9

7

1

9

9

8

1

9

9

9

2

0

0

0

2

0

0

2

2

0

0

3

2

0

0

4

Schweiz Med Wochenschr (18712000)

Swiss Med Wkly (continues Schweiz Med Wochenschr from 2001)

EditoresMedicorumHelveticorum