Archives of Disease in Childhood 1994; 70: F107-F 11 F

Lipid peroxidation as a measure of oxygen freeradical damage in the very low birthweight infant

T E Inder, P Graham, K Sanderson, B J Taylor

AbstractOxygen free radical mediated tissueinjury is implicated as a major factor inthe pathogenesis of the long term com-plications seen in the premature infant,and direct evidence of their role in thedevelopment of these long term problemsis lacking. A prospective observationalstudy of 78% of very low birthweightinfants admitted to a level III neonatalintensive care unit in 1992 was undertakento determine the relationship betweenlipid peroxidation products, antioxidantactivity, and outcome. Lipid peroxidation(malondialdehyde-thiobarbituric acid,MDA-TBA) and antioxidant activity(vitamin E and glutathione peroxidaseactivity) were measured in 22 very lowbirthweight infants in the cord blood andthe infant's blood at 24 hours, 48 hours,and 1 week ofage and correlated with out-come measures. The normal range forthese measures was established in thecord blood samples of 48 consecutivehealthy full term infants.The concentration of MDA-TBA at 1

week correlated with the number of daysof oxygen treatment and number of daysof positive pressure ventilatory support.Controlling for gestational age and ante-natal complications simultaneously theMDA-TBA concentration remainedsignificantly associated with the numberof days of oxygen treatment and thenumber ofdays ofpositive pressure venti-latory support. Glutathione peroxidasewas low in the premature and full terminfants consistent with the low concen-trations of selenium known to be presentin southern New Zealand. There wasevidence of a quadratic relationshipbetween vitamin E at 1 week and the totalnumber of days of supplementary oxygenrequirement, with both high and lowvalues associated with increased oxygenrequirement. This association, however,did not remain after controlling for gesta-tional age and antenatal complications.These results support the role of oxygenfree radicals in mediating tissue damageassociated with the development ofchronic lung disease in the prematureinfant.(Arch Dis Child 1994; 70: F107-1 1 1)

Free oxygen radicals are extremely reactivespecies that although crucial to a wide range ofnormal biological processes, are potentiallydamaging. They can disrupt lipid cell

membranes, destroy cell enzyme functions,alter DNA, and lead to cell death.' 2 Freeoxygen radicals appear to have a role inmediating tissue injury in hyperoxia,l-4 indamage associated with reperfusion afterischaemia, and in inflammatory processes. 1 2 5 6These are all mechanisms implicated in thepathogenesis of the major complications of pre-maturity including bronchopulmonarydysplasia, retinopathy of prematurity, andintraventricular haemorrhage.7-9

Oxygen free radical damage is usually pre-vented by a series of enzymatic defences such assuperoxide dismutase, glutathione peroxidase,and catalase and by a series of non-enzymaticquenching antioxidants such as vitamins E, A,and glutathione.' The premature infant haspoorly developed antioxidant systems andtherefore may be at increased risk of radicaldamage.7 8 1012 This risk may be even greaterfor infants in New Zealand where environmen-tally low selenium concentrations may result inlow activities of the selenium dependentenzyme glutathione peroxidase. 13 14A major limitation in this area of research

has been the absence of an accurate andreliable measure of free radical activity.Oxygen free radicals react with lipids to pro-duce lipid peroxide products.2 Measurementof lipid peroxide concentrations by themalondialdehyde-thiobarbituric acid (MDA-TBA) assay is the most widely recognisedmeasure of free radical activity used in currentclinical research.'5 16 Free radical activity inthe very low birthweight infant has beenassessed by expired ethane and pentane,8 butthere are no reports of attempts to measurelipid peroxidation products and antioxidantactivity in the sera of premature infants andcorrelate these concentrations with outcome.

Patients and methodsSerum concentrations of MDA-TBA andvitamin E and glutathione peroxidase activitywere measured in samples of (i) cord bloodand (ii) infant's blood at 24 hours, 48 hours,and 1 week of age in 22 very low birthweightinfants admitted to the neonatal intensive careat Dunedin Public Hospital. All infants lessthan 1500 g and/or less than 32 weeks'gestation were eligible to participate. Of allvery low birthweight infants born during thestudy period October 1991-November 1992,78% were enrolled. For comparison, cordblood samples from 48 full term infants wereanalysed.

Full details of the pregnancy, birth history,and neonatal course including details ofnutrition, blood transfusions, oxygen supple-

Dunedin Hospital,Dunedin, NewZealand, DepartmentofPaediatrics andChild HealthT E InderB J Taylor

Department ofSurgeryK Sanderson

Department ofCommunity Healthand General Practice,Christchurch School ofMedicine, Universityof Otago, Dunedin,New ZealandP Graham

Correspondence to:Dr B J Taylor, Departmentof Paediatrics, University ofOtago, PO Box 913,Dunedin, New Zealand.Accepted 1 1 October 1993

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mentation, and ventilatory support werecollected prospectively. Ultrasound scans ofthe head were performed in the first 48 hours,at 1 week, and 6 weeks of age. Screening forretinopathy ofprematurity was undertaken at 6weeks of age by a paediatric ophthalmologist.Follow up of neurodevelopmental outcomecontinues to be collected. All infants in thestudy were treated in accordance with standardprotocols in the neonatal unit. Oxygenationwas monitored by continuous pulse oximetry(Nelcor N100 and N200, target oxygen satura-tion 87-95%) and by frequent blood gasanalysis. Arterial oxygen tension was main-tained at 6-0-10-0 kPa. All infants werereceiving parentally administered glucose andsix (27%) infants also received enteral feedingfrom the second day onward. Total parenteralnutrition (TPN) was commenced on the thirdday in 17 (78%) infants using 1 g/kg/dayTrophamine (Kendall McGaw Laboratories,USA) with 10-15% dextrose. Intralipid (KabiPharmacia) was commenced in 14 (64%)infants on day 3-4 at 1 g/kg/day. The aminoacid mixture and the lipid were both increasedas tolerated by 0 5 g/kg/day to a maximum of 3g/kg/day. Supplementary trace elements ofzinc, copper, manganese, iodine, chromium,molybdenum, and selenium at 1-4, 0-6, 0 04,0-05, 0 005, 0-002, and 0 04,umol/kg/day wereroutinely added to the TPN solution.Supplementary vitamins were added to theTPN solution as MVI Paediatric (Rhone-Poulenc Rorer) 1 1 ml/kg/day giving 506 USPunits/kg/day vitamin A, 88 USP units/kgldayvitamin D, and 1-54 USP units/kgldayvitamin E.

SAMPLE ANALYSISMDA-TBA was measured in plasma after lipidextraction by reverse phase high performanceliquid chromatography (HPLC) using aShimadzu RF-530 HPLC fluorescence detec-tor (excitation 515 nm, emission 553 nm)coupled to a Shimadzu Chromatopac C-R3Aintegration. Intra-assay and interassay vari-ability were 3 and 5% respectively.17 18

Vitamin E was measured by HPLC methodof Bieri19 with intra-assay coefficient ofvariation less than 2%.

Glutathione peroxidase was measured witht-butyl hydroperoxide as a substrate by amodification of the method by Paglia andValentine.20 Each run was standardised againsta stored haemolysate with intra-assay andinterassay variability of 2% and < 10% respec-tively.

Results are reported as mean (SD) unlessspecifically described otherwise.

CHARACTERISTICS OF THE STUDY GROUP

MDA-TBA, glutathione peroxidase, andvitamin E were measured in the cord sera of 48consecutive healthy full term infants with meanbirth weight 3320 (420) g (range 2480-4860g) with mean gestation 39-4 (0 8) weeks (range37-43 weeks). MDA-TBA, glutathioneperoxidase, and vitamin E were analysed in

serum samples of 22 very low birthweightinfants from cord blood and the infant's bloodat 48 hours and 1 week. Fifteen of these infantsalso had sera analysed at 24 hours of age. Ofthe 22 very low birthweight infants, 13 wereboys and nine girls. The mean gestation was27-9 (2-2) weeks (range 24-32 weeks). Themean birth weight was 1080 (253) (range580-1480 g).The presence of chronic lung disease was

defined as supplementary oxygen requirementfor greater than 28 days with an abnormalchest radiograph and respiratory status.

ETHICSThe study protocol was approved by the ethicscommittee of Otago Area Health Board andwritten consent was obtained from the parentsto allow participation of their infants.

STATISTICAL ANALYSISConventional analysis of variance was used forbetween group comparison of normally dis-tributed variables while the Kruskal-Wallisnon-parametric test was used for variableswhich were clearly not normally distributed.Fisher's exact test was used for between groupcomparisons of dichotomous variables. Thecord and postnatal concentrations of MDA-TBA and vitamin E and glutathione per-oxidase activities and their ratio were analysedin relation to days of ventilator and oxygendependence by linear regression. For ease ofinterpretation regression coefficients wereconverted to correlation coefficients. Theinfluence of gestational age, sex, and antenatalvariables on these correlations were analysedvia a multivariate linear regression model.

ResultsVery low birthweight infants who developedchronic lung disease were of earlier gestation(F=3-93, p=0 07) and lower birth weight(F=7 09, p=0 02), with greater maximumoxygen requirement in the first 12 hours(F=6-83, p=0 03). Antenatal factors did notdiffer significantly between the outcomegroups (table 1).

Table 1 Characteristics of infants with chronic lungdisease and with normal respiratory outcome

Chronic Normallung respiratorydisease outcome(n= 16) (n= 6)

Gestational age (weeks) 27-3 (2.2) 29-3 (1-8)Birth weight (g) 1000 (230) 1290 (190)Sex (male:female) 10:6 4:2Antepartum haemorrhage 2/16 3/6Prolonged rupture of membranes 7/16 5/6Pre-eclampsia 4/16 0/6Elective delivery 11/16 6/6Maximum fractional inspired

oxygen in first 12 hours (%) 47-5 (18) 23-5 (3)Death 0/16 0/6Days ventilated 18-3 (12-6) 1-7 (2-3)Days in oxygen 65-8 (32-9) 4-8 (2 9)Oxygen at term 8/16 0/6Retinopathy 0/16 0/6Intraventricular haemorrhageGrade 1-2 1/16 0/6Grade 3-4 1/16 0/6

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Table 2 Mean (SD) MDA-]blood offull term andprematurlung disease

MDA-TBA (j±mol/1)Vitamin E (1Lmolfi)*Glutathione peroxidase (U/1)

*Vitamin E conversion factor: j.n

rBA, vitamin E, and glutathione peroxidase in the cord In the cord blood samples of the prematuree infants who subsequently did or did not develop chronic infants who developed chronic lung disease the

concentration of MDA-TBA was higherPremature infants (Kruskal-Wallis test, X2=27-5, p=0 0001), the

Full term Chronic lung Chronic lung concentration of vitamin E lower (Kruskal-infants disease absent disease present Wallis test, X2=1795, p=0000O1), and gluta-(n=48) (n=6) (n=16) thione peroxidase activity lower (x2= 11 5,630 (94) 71-6 (9-2) 85-3 (17-5) p=0-003) than the healthy full term infants89 (19) 74 (21) 56 (28) (table 2). These values were not influencedby8175(17) maternal age, previous obstetric history,

nol/l=mg/lX2 32. complications during the pregnancy, mode ofdelivery, antenatal steroid use, duration oflabour, or Apgar scores at 1 or 5 minutes.The concentrations of MDA-TBA and

vitamin E and glutathione peroxidase activity200 _ Chronic lung at each sample time postnatally did not cor-

disease ** relate with the simultaneous measurements of

o Present _| ventilation status, oxygen requirement, form of150 nutrition, or transfusion volume.

For infants who developed chronic lungo disease the mean MDA-TBA concentrationsE_L T//-werehigher with the mean vitamin E and< 100 glutathione peroxidase values lower than those

infants without chronic lung disease (figure).0 f Preliminary analysis suggested that the cord,E

50day 1, and day 2 measurements were not

50 related to the total number of days of oxygen

treatment.Regression analysis of the concentration

o of MDA-TBA at l week showed a strongCord 1 2 3 4 5 6 7 correlation with the total number of days

120 positive pressure ventilatory support (r=0-54,p=0009) and the total number of days ofoxygen treatment (r=0-61, p=0 003). Therewas evidence of a quadratic relationship

/ \t between vitamin E at 1 week and the totalnumber of days supplementary oxygen treat-u80-v gj --- X ment, with both low and high concentrations

o associated with increased oxygen requirement.X v (For the quadratic model R2=0 34, p=004,

while for the linear model R2=0-02, p=0 53. As 40 _ comparison of the two models based on the

reduction in error sum of squares yielded_ F=7-7, p=0-017.) Vitamin E at 1 week also

showed evidence of a quadratic relationshipwith days of positive pressure ventilatory

0C 3 4 5 6

support, although the strength of the relation-Cord 1 2 3 4 5 6 7 ship was not as strong as that found in relation

30 to the number of days of supplementaryoxygen. (R2=0-27, p=0 09 for quadraticmodel; R2=0.015, p=0-63 for linear model.

* z Fit of quadratic significantly better F=5-207,p=0 037.) The activity of glutathione per-

_ 20 oxidase activity at 1 week was weakly inversely0/ related to the total days of positive pressure

E / ventilatory support (r=-0 398, p=0 08) butWn / : did not correlate with the total number of days.' of supplementary oxygen treatment (r= - 0 05,. lo - p=083).> The association between outcome measures

and MDA-TBA, vitamin E, and glutathioneperoxidase was investigated further by con-

I_____I____I_____I____I_____I____I____I_ trolling for the potentially confounding factors0

6 7via multiple linear regression. The con-

Corg1e 3f4nfant 5da 6founders controlled were gestational age,and the presence or absence of antepartum

Vaniations in plasma values ofMDA-TBA, glutathione haemorrhage, prolonged rupture of mem-peroxidase, and vitamin E in premature infants with and branes, and pre-eclampsia. Controllingwithout chronic lung disease; values are mean (SE); simultaneously for these factors slightly*p<O1, **p<005. Vitamin E conversionfactor:,umoIl=mg/lX2-32. strengthened the positive association between

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MDA-TBA and the number of days of supple-mentary oxygen requirement (partial r=0-66,t=3-57, p=00026) and between MDA-TBAand the number of days of positive pressureventilation (partial r=058, t=2-85,p=000115). After controlling for the poten-tially confounding factors glutathione per-oxidase was not associated with either totaloxygen requirement (partial r=0-26, t= 1 -036,p=0-318) or with the number of days of posi-tive pressure ventilation (partial r=012,t=-0 477, p=0641). Vitamin E did notremain significantly associated with oxygenrequirement (F=1-33, p=030) or days ofpositive pressure ventilatory support(F= 1 0102, p=0 366) after controlling for thesepossible confounders.

DiscussionOur results demonstrate a correlation of theconcentrations of MDA-TBA with respiratoryoutcome in the very low birthweight infant.The small numbers in our study limit the con-clusions relating to glutathione peroxidase andvitamin E. MDA-TBA is the most extensivelyused measure of lipid peroxidation in currentclinical research.'5 16 21 Individual researchlaboratories have refined their own assaysmaking comparisons between values, methods,and investigations difficult.15-17 21 The assayhas been criticised as a measure of radicalactivity and resultant lipid peroxidation as it isindirect and can be contaminated by otherthiobarbituric acid reactive species. Thesesources of error have been reduced in our assayby the addition of butylated hydroxytoluene tolimit intra-assay production ofMDA-TBA andthe use of lipid extraction and ion exchangehigh pressure chromotography to removeinterfering chromogens.17 18MDA-TBA is not a sensitive measure of

radical activity requiring high levels of radicalactivity to alter its concentration.2' The findingof significant increases in MDA-TBA indicatesthat the infants who developed chronic lungdisease were exposed to very high levels ofradical activity. The increase in cord concen-trations of MDA-TBA in the prematureinfants who later developed chronic lungdisease may be an explanation of the wellknown vulnerability of premature infants tomaternal factors. Mild increases in lipid per-oxidation (MDA-TBA) have been foundbefore delivery in all women, but with womenwith complicated pregnancies (pre-eclampsia)significantly higher concentrations of lipid per-oxides and significantly reduced antioxidantactivity have been reported.22 23 Our resultssupport the hypothesis that maternal factorsbefore delivery increase the vulnerability of thepremature infant to long term complicationsby increased free radical activity and a reduc-tion in antioxidant protection.The antioxidant status of the premature

infant has been shown to be poorly devel-oped.7 10A2 Antioxidant enzymes maturethroughout gestation. Low antioxidant enzymeactivity in the premature infants' lungs poorlyequip them for assisted ventilation in a hyper-

oxic environment.47 Antenatal administrationof glucocorticoids matures the antioxidantenzyme system,24 whereas antenatal thyro-trophin releasing hormone impairs antioxidantenzyme maturation.25 26 Surfactant replace-ment treatment has produced only limitedimprovement in the rates of chronic lungdisease.27 28 The role of the antioxidantenzyme system in the onset and progression ofhyperoxic lung injury in premature experimen-tal animals suggest that the premature infantmay benefit from therapeutic intervention withantioxidant enzyme preparations.' 029-31Rosenfeld et al evaluated the effect of subcuta-neous superoxide dismutase in a randomisedblinded controlled study of 45 infants withsevere respiratory distress syndrome (ventilatordependent with fractional inspired oxygen>0 7 at 24 hours) administered until infantswere in room air without ventilator support.32Radiological and clinical signs of broncho-pulmonary dysplasia were significantlyreduced in surviving infants who receivedsuperoxide dismutase (3/14 treated v 11/17non-treated). There was no difference inthe number of days of oxygen dependence inthe two groups. The therapeutic use of antiox-idant enzyme preparations requires furtherresearch.

Vitamin E has multiple antioxidant func-tions that allow the quenching of free radicals,particularly the superoxide radical.33 Thesuperoxide radical is an important destroyer ofpolyunsaturated fatty acid components ofbiological membranes. Newborn infantsusually receive high doses of vitamin E incolostrum producing the marked increase (3-4fold) in vitamin E concentrations in the firstweek of life.34 35 Our results suggest that theinfants who developed chronic lung disease didnot experience the anticipated rise in vitamin Econcentrations during their first week of life.This was not explained by differences in feed-ing patterns. Premature infants may requirehigher doses ofvitamin E to maintain adequateserum concentrations.36 Our results suggestedthat infants in our study with both low, that is< 11 7 ,umol/l (<5 mg/l) and high, that is >28pumol/l (> 12 mg/l) vitamin E concentrations at1 week were at high risk for development ofchronic lung disease. These infants receivedvery similar intakes of vitamin E in their firstweek and it is hypothesised that the infantswith raised plasma concentrations may bemobilising vitamin E from their tissues,increasing oxidant vulnerability at the tissuelevel.

Glutathione peroxidase requires selenium asan essential component. New Zealand, partic-ularly in the southern region, is selenium defi-cient and low glutathione peroxidase activityhas been shown in adults and infants in thisregion.'3 14 Very low birthweight infants in thisstudy had some of the lowest glutathioneperoxidase activities reported. Glutathioneperoxidase is a protective antioxidant enzymeand the reduced activity in these infantswill increase their vulnerability to radicaldamage.29 30 The inability to show anysignificant difference between activities of

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glutathione peroxidase in relation to respira-tory outcome in our study may be due to thefact that the values are uniformly extremelylow. New Zealand has a high incidence ofbronchopulmonary dysplasia, not explainedpurely by a high survival rate, in premature

infants.3' Selenium deficiency with resultinglow glutathione peroxidase activity may be a

factor increasing vulnerability for New Zealandinfants to radical mediated tissue injury.

This investigation shows that in very lowbirthweight infants the probability of chroniclung disease increases with documented freeradical activity evidenced by increased lipidperoxidation products. The role of the anti-oxidants vitamin E and glutathione peroxidasein the vulnerability of these infants requiresfurther investigation. Demonstration of thecausative role of free radicals requires inter-vention studies with the possibility of usinglipid peroxidation as a measure of the effective-ness of the antioxidant intervention. MDA-TBA appears to be a useful measure inexploring the role of free radicals in thediseases of the premature infant.

The authors would like to thank Vicki Phillips, Glenys Taylorfor their dedicated work with the MDA-TBA assays in theseinfants. We would also like to acknowledge the financialsupport of the Grand Lodge of New Zealand Freemasons andthe New Zealand Lottery Board. Mr Graham was funded by theHealth Research Council ofNew Zealand.

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