Turkish Neurosurgery 3: 150 - 153. 1993 Anaoglu: Lipid Peroxidation in Brain Tumour

Investigation of Serum and Tissue Lipid Peroxidation andSerum Ascorbic Acid, and Iron Levels in Human Brain Tumour

AYSEL ARICIOGLU. SÜKRÜ AYKOL. ETHEM YENIKAYA, KEMALI BAYKANER.M. SERDAR ALP. NURTEN TÜRKÖZKAN

Department of Biochemistry (AA. EY. NT) and Neurosurgery (SA. KB. MSA). School of Medidne. Gazi University. Ankara. Türkiye

Abstract , The present studyaimed the biochemical relationshipsbetween human brain tumours and serum lipid peroxide. ascor­bic add and total iron levels. Tissue lipid peroxide levels in tumourtissue itself and in the surrounding tissue were also measured.Serum lipid peroxide levels were found to be significantly high(p<O.OOI)in patients with brain tumour while the serum ascorbicadd levels were significantly low (p<O.OOI)compared with the con­trol group. Serum iron concentrations in patients with braintumours were also found to be high but the results were nor

INTRODUCTION

There is inereasing evidenee to suggest that theinerease of lipid peraxidation in membranous strue­tures eauses many pathologieal states in humans bydamaging the membranes and interlering with thefunetion of organelles and tissues (20.21.25.28). Bio­membranes and subeellular organalles are partieular­ly sensitive to oxidative attacks as they earry speci.fieamounts of polyunsaturated fatty adds (PUFA) intheir membrane phospholipids (15.20.21.30).

In reeent years. lipid peraxidation in tumour eellshas been investigated by several research groups (2.3).Malignant tumours and regenerating tissue exhibit alaw degree of "peraxidizability" that has been shownto be ralated to the growth rate of the tumour (4.7.14).

Altered lipid eomposition of the eellular membraneswhich have deaeased PUFA eauses important chan­ges in the static and dynamie praperties of the mem­branes and is assodated with law suseeptibility toperaxidation. Cellular oxy-radica1scavenging enzyrnes

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statistically significant. On the other hand lipid peroxide levelsin the tumour tissues were significantly lower than in the surroun­ding tissue (p<O.OOI).We believe that high serum lipid peroside levels in patients withbrain tumours are the result of decreased serum ascorbic add con­

centrations and the pressure effeet of tumours on surroundingtissues which cause lipid peroxide levels in periphera! tissues andin the serum to increase.

Key words, Ascorbic add. Brain turnour. Iron. Lipid peroxidation.

are markedly redueed (ll). Lipid peraxidation can beindueed by iran ions as reported by Kappus (10). Inprevious studies the interrelatian of aseorbie add andiran with lipid peraxidation has been investigated ex­perimentally within th~ tissues (13.15). Lack of infor­mation regarding the levels of lipid peraxide in serumand brain tumour tissues and alsa the serum iran andaseorbie add level interrelatian with brain tumours

direeted us to investigate this area. In this study weexamined the serum aseorbie add and iran levels of

patients with brain tumours and measured serum andtissue lipid peraxide levels in these patients.

MATERIALS AND METHOD S

This study was earried out on 30 patients withbrain tumours between the ages 20-65. The controlgraup included 30 healthy people with similar agevariations. Brain tumours and surraunding tissueswhich had been removed surgieally were washed insaline solution and homogeriized with cold 1.15% KClto make 10% homogenates.

Patients

DlSCUSSION

Brain tumoui tissue (n= io)Surrounding tissue (n= io)Significancea Means + SEM

Turkish Neurosurgery 4: 150 - 153. 1993

Tissue lipid peroxide was determined using themethod of Dchiyama and Mihara (24). Briefly. 3 ml1%phosphoric acid and 1ml 0.5%thiobarbituric acid(TBA)were added to a 0.5 ml homogenate and themixture was kept in a water bath a 95 C for 45minutes. The caloured reaction product was ex­tracted with 4 ml n-butanol and the difference of ab­sorbances at 535 and 520 nm was recorded. The

breakdown product of 1.1.3.3 tetraethoxypropanewas used as standard and the values were express­ed as nmol malondialdehyde / gram (MDA/g) tissue.

Serum lipid peroxide levels were measured us­ing the method desaibed by Yagi (27). 20 ul serumwas mixed with 4 ml N/12 sulphuric acid and then0.5 ml 10% phosphotungstic acid was added. Afterstanding at room temperature for 5 minutes. the mix­ture was centrifuged at 3000 rev/min for 10 minutes.The supernatant was discarged and the sedimentwas mixed with 2 ml N/12 HiS04 and 0.3 ml 10%

phosphotungstic acid before centrifugation wasrepeated. The sediment was suspended in 4 mldistilled water and then 1ml thiobarturic acid (TBA)reagent (a mixture of equal volumes of 0.67% TBAaqueous and glacial acetic acid) was added andheated the 95 C for 60 minutes in an oil bath. After

caoling with tap water. 5.0 ml n-butanol was addedand the mixture was shaken vigorously. After cen­trifugation an 3000 rev/min for 15 minutes. the n­butanol layer was taken for gluorometric measure­ment at 553 nm with excitation at 515 nm. By takingthe fluorescence intensity as f and that of the stan­dard solution. obtained by reacting 0.5 nmtetramethoxypropane with TBA.as F. the lipid perox­ide (Lp) level was expressed in terms of malon­dialdehyde: Lp:0.5.f/F.1.0/0.02=f/F.25(nmollml serum)

Serum ascorbic acid (AA) concentrations weredetermined using a modified 2,4-dinitrophenylhydra­zine method (17). The results were expressed asmilligrams AA/dI.

Serum iron was determined using the proteinpredpitation method; values were expessed in ugr/d (9).

The studenfs t test was employed for statisticalcomparison of the values.

RESULTS

The serum lipid peroxide. ascorbic acid and ironlevels for patients with and without brain tumour areshown in Table i.

Anaoglu: Lipid Peroxidation in Brain Tumour

Results of peroxide values of brain tumour andsurrounding tissue are shown Table II.

Tablo i :Comparison of Serum Lipid Peroxide. AscorbicAcid and Iran Levels of Patients With and

Without Brain Tumours.Patients Lipid Perolide Ascorbic AcidIron

(nmoUm!)

(mg/d!)(ug/d!)

With tumaurs (0=30)

7.28+1.99a0,32+0.09595.27 +

!1.58 Withaul tumaurs (0= 30)

3.66§0.580.69+01191.50+

10.46 Significance

p<O.OO!p<O.OO!p<0.05

a Means + SEM

Table II: Comparison Between Levels of The Lipid Peroxideof Tumour Tissue and Surrounding

Tissue. in Patients With Brain Tumours

Lipid Peroxide(nmollg.tissue)

17.80+89.4a59.00+24.43

p<O.OOI)

Free radicals are formed during the normalmetabolism of aerobic cells and free radical damagemay cause some pathological states by either enhan­cing the production of the radicals or deaeasing theantioxidative deferce mechanisms of the tissues

(12.20.21.22).

Free oxygen radicals and lipid peroxides are short­lived products and difficult to measure directly.Malondyaldehyde on the other hand. is a more stableand long-lived degradative product of lipid peroxideand is often assayed as reflecting lipid peroxidationlevel (13.22).

When the fored lipid peroxides accumulate to acertain degree they leak from the tissue into thebloodstream and cause an inaease in lipid peroxidelevels in the serum. Accarding to Yagi. the blood lipidperoxide level often carrelates with the severity ofthe disease (27).

In this study. we compared serum and tissue lipidperoxide levels with brain tumours and found thatthe serum lipid peroxide levels of patients with braintumours were significantly higher than those of thecantrol group (p<0.001). The serum iron levels were

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Turkish Neurosurgery 4: 150 - 153, 1993

also found to be high in these patients though theresults were not statistically signincant.

Previous studies have conduded that lipid perox­idation is signincantl deaeased in tumour cells andsurrounding tissues compared with the correspon­ding normal tissues (3).Peroxidative activity appearsto be inversely related to the growth rate of thetumours. It is suggested thad the low susceptibilityof tumour membranes to peroxidative agents maybe a factor responsible for the high mitotic activityof these tissues (1.4).

lron plays an important role in the biochemistryof oxygen spedes. it catalyses the Fenton reaction.can react with oxygen or superoxide anion and canpartidpate in chain propagation lipid reactions BothFe(II)and FE(III)are important in these processes. Ex­tracellular iron is complex (transferrin. lactoferrin.haemoglobinlhaeme) although free iron can be releas­ed at low pH. The brain contains large amounts offerritin which is distributed heterogeneously (12.19).

Rehnaona et al investigated the peroxidation of cor­tical phospholipids in vitro using Fe+2 and ascorbicadd stimulation (16).According to their reporte Fe+2

is an important pro-oxidant within the system.

Watson et aL. reported the assodation of freeradical process es with incomplete brain ischaemia incat brain using decreased ascorbate levels as an in­dicatar (26). In their report Flamm et aL. founddecreased tissue concentration ofascorbic add dur­

ing prolonged incomplete brain ischamia and daim­ed that this consumption of naturally occurringscaverger was due to pathological free radical reac­tions (6).

it is now well established that oxygen freeradicals are generated in the ischaemic reperfusedtissues (5.S. 1S,29).

Consequently. we believe that in patients withbrain tumours the pressure caused by tumour tissueresults in ischaemia of the surrounding tissue whichcauses an inaease in lipid peroxidation and serumlipid peroxide level.

Correspondence: Sükrü AkyolDepartment of NeurosurgeryGazi University Medical SchoolBesevler. 06510, Ankara

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Anaoglu: Lipid PeroxidatIon in Brain Tumour

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