British Journal of Obstetrics and Gynaecology August 2000, V01107, pp. 982-986

Lipid peroxidation in umbilical arterial blood at birth: the effects of breech delivery

*Hakan Kaya Associate Prpfessor, *Baha Oral Associate Professor, TRalf Dittrich Assistant Professor, *Okan Ozkaya Research Assistant (Obstetrics and Gynaecology)

*Siileyman Demirel University, School of Medicine, Department of Obstetrics and Gynaecology, Isparta, Turkey; tDepartment of Obstetrics and Gynaecology, Friedrich-Alexander University, Nuremberg-Erlangen, Germany

Objective To determine oxygen free radical activity in breech presentation at birth and relate it to umbil- ical cord blood acid-base status.

Design A series of 63 singleton deliveries (28 cephalic deliveries as controls), 23 breech deliveries with normoacidemia, and 12 breech deliveries with mild acidaemia) had determination of malondialde- hyde and acid-base parameters.

Setting The delivery suite in the Department of Obstetrics and Gynaecology at the Suleyman Demirel University, Isparta, Turkey.

Participants Sixty-three singleton infants born at term with spontaneous or induced labour and initially normal fetal heart rate tracing.

Methods After delivery, umbilical cord arterial and venous blood samples were collected for the deter- mination of malondialdehyde concentrations. Oxygen saturation, PO,, pC02, pH, and base excess were also measured.

Main outcome measures Umbilical cord arterial and venous blood gases and malondialdehyde levels.

Results There was a significant correlation between umbilical arterial and venous levels of malondi- aldehyde and all acid-base parameters (P < 0.001). There were negative correlations between malon- dialdehyde levels and pH, PO, and bicarbonate, while there was a positive correlation between malondialdehyde concentrations and pC02. A positive correlation between malondialdehyde levels and base excess was present in the control group and total breech group (n = 35). The malondialde- hyde levels in the total breech group, nonacidaemic breech group (n = 23) and the mildly acidaemic breech group (n = 12) were significantly higher than those in the control group (P < 0.0001). How- ever, acid-base parameters in the nonacidaemic breech group were not statistically different from those in the control group.

Conclusion Lipid peroxidation products (malondialdehyde) existed to some extent in the umbilical cords of newborns with normal acid-base parameters in breech delivery. Our data support the con- tention that lipid peroxide may be a more sensitive measure for metabolic dysfunctions due to fetal hypoxia than acid-base balance.

INTRODUCTION The incidence of breech presentation varies with fetal maturity. Breech delivery has some disadvantages over cephalic delivery. Because of the irregular outline of the breech, spontaneous rupture of the membranes may be followed by cord prolapse. This is not always as seri- ous as in a cephalic presentation because the soft breech is less likely to compress the cord, although the cord may still go into spasm and cause acute fetal hypoxia. In addition, the volume of the uterus decreases

Correspondence: Professor H. Kaya, SDU Tip Fakiiltesi, Kadin Hastaliklan ve Dogum ABD, Isparta, Turkey.

after the delivery of the fetal body, which consequently reduces the area of the placental site, and increases the fetal hyp~xia l -~ .

Perinatal hypoxia is known to be one of the major causes of perinatal morbidity and mortality4. Persistent intrauterine hypoxia may result in ischaemia, leading to permanent damage of the brain. Hypoxic insults to tis- sues cause the formation of oxygen-derived free radi- cals, which may induce brain injury in a hypoxic infants-'. Free radical generation causes peroxidation of unsaturated fatty acids resulting in increased levels of aldehydic lipid peroxidation products such as malondi- aldehyde, which can be measured by the thiobarbituric acid reactions-lO. In this study, we investigated lipid

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LIPID PEROXIDATION IN UMBILICAL ARTERIAL BLOOD 983

peroxidation in the term breech fetus without any complications or with acidaemia, as a measure of oxidative stress at birth.

METHODS This study included 28 pregnant women with a cephalic presentation (Group A, control group) and 35 women with a breech presentation, all having singleton preg- nancies, between 37 and 42 weeks of gestation and delivered vaginally in the Siileyman Demirel University Hospital, Isparta, Turkey between February 1995 and December 1996. Women who had elective or emer- gency caesarean section were excluded. The study was approved by the Suleyman Demirel University of Isparta ethics committee, and all women gave pennis- sion for the study. None of the women suffered from medical diseases or had evidence of fetal distress prior to recruitment.

The 35 women with a breech presentation were divided into two further groups: Group B, 23 cases with umbilical arterial pH > 7.20, and Group C, 12 cases with umbilical arterial pH I 7.20.

Induction of labour was performed in 10 women with a cephalic presentation, six women with breech presentation with umbilical arterial pH > 7.20, and two women with breech presentation with umbilical arte- rial pH I 7-20. None of the women received prostaglandins for induction of labour. Immediately after delivery, a segment of umbilical cord was double clamped, and blood was drawn from the umbilical artery and vein into separate 10 mL preheparinised plastic syringes. The whole blood samples were anal- ysed within 5 minutes of collection for pH, pCO,, PO,, base excess and bicarbonate (ABL 505 Radiometer, Copenhagen). The plasma in both the arterial and venous blood samples was collected immediately after refrigerated centrifugation at 1000 revolutions per minute for 10 minutes, and then stored at -20°C before the malondialdehyde assay was undertaken.

The plasma malondialdehyde levels were estimated as reactive substances by a thiobarbituric acid adduc- tion method described by Wasowicz et aL8. Fifty pL plasma samples were introduced into tubes each con- taining 1 mL distilled water. After addition of 1 mL of a solution containing 29 mmoVL thiobarbituric acid (Sigma Chemical Co, Deisenhofen, Germany) into acetic acid (8.75 mmoVL, pH of the reaction mixture, 2.4-2.6), the samples were placed in a water bath and heated for 1 hour at 95-100OC. After the samples had cooled under running cold water, 25 pL of 5 molVL hydrochloric acid were added, the reaction mixture was extracted with 3.5 mL of n-butanol (Sigma Chemical Co, Deisenhofen, Germany) and centrifuged at 3000g per minute for five minutes. After centrifugation, the

butanol phase was separated and the fluorescence of the butanol extract was measured in a spectrofluorometer (Schimadzu-RF-5000, Kyoto, Japan), using 525 nm for excitation and 547 nm for emission.

Statistical analysis was performed using SPSS Ver- sion 6.1 for Windows. Correlation coefficients were estimated using Spearman’s rank correlation test. Mal- ondialdehyde levels in the three groups were compared by the Kruskal-Wallis test. The three groups were then compared in pairs using the Mann-Whitney U test.

RESULTS The women’s ages ranged from 21 to 36 years (mean 26, SD 4) in Group A, 19 to 37 years (mean 25, SD 6) in Group B, and 18 to 33 years (mean 25, SD 5 ) in Group C. The mean (SD) gestational ages in the groups were 39 (1) weeks, 40 (1) weeks and 40 (1) weeks, respec- tively. There were no statistically significant differences between the groups as regards age, gestational age, birthweight and antenatal complications.

The mean (SD) umbilical arterial malondialdehyde levels were 151 (0-12) nmoVmL, in Group A 1.74 (0.1) nmoVmL in Group B and 1-92 (0.16) nmoVmL in Group C. The mean (SD) umbilical arterial pH values were 7.28 (0-03) in Group A, 7-27 (003) in Group B and 7.17 (0.02) in Group C. A significant positive cor- relation (Pearson) existed between arterial and venous levels of malondialdehyde, pH, pCO,, PO,, bicarbon- ate, and base excess. The mean arterial levels were higher than the venous levels for malondialdehyde, pCO,, and bicarbonate but lower for pH, PO, and base excess (Table 1).

The mean (SD) duration of labour was 7.07 hours (2.6) in Group A, 10.04 hours (1-7) in the Group B and 10-66 hours (1.6) in the Group C. The number of infants who had an Apgar score of I 6 at 5 min was 1/28 in Group A, 11/23 in Group B and 8/12 in Group C. Arterial malondialdehyde levels had a significant negative correlation with umbilical arterial pH, PO,, bicarbonate and base excess, whereas it showed a sig- nificant positive correlation with pC02. There was no significant correlation between umbilical arterial mal- ondialdehyde levels and the duration of labour in all three groups (Table 2).

Arterial plasma levels of malondialdehyde were sig- nificantly higher in the nonacidaemic breech deliveries and total breech deliveries than cephalic deliveries (P c 0.0001). There was also a significant difference between the acidaemic breech deliveries and nonaci- daemic breech deliveries (P = 0-0025) (Table 3).

The relationships between umbilical arterial pH, base excess and malondialdehyde concentration according to cephalic and breech deliveries are shown in Figs. 1 and 2.

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984 H . KAYA ET A L .

Table 1. The levels of malondialdehyde (MDA), pH, pCO,, p02. bicarbonate and base excess in the umbilical artery and the umbilical vein, with the arterio-venous differences by observational group. Values are given as mean (SD). A = cephalic control group; B = nonacidaemic breech group; C = acidaemic breech group.

Parameter measured Arterial Venous Arterio-venous difference

MDA (nmoVmL) A 1.51 (0.12) 1.41 (0.11) 0.089 (0.032) B 1.74 (0.10) 1.66 (0.12) 0.087 (0.034) C 1.92 (0.16) 1436 (0.18) 0.027 (0.044)

A 7.28 (0.03) 7.31 (0.02) -0.027 (0.007) PH

B 7.27 (0.02) 7.29 (0.02) -0.022 (0.008) C 7.17 (0.02) 7.19 (0.02) -0.026 (0.009)

PC02 ( m n w 1 1.464 (1.770) A 50.07 (6.65) 38.60 (6.62)

B 48.82 (6.41) 36.69 (7.13) 12.130 (2.001) C 61.92 (8.40) 53.33 (8.77) 8.583 (3.507)

Po, A 20.28 (3.91) 29.75 (4.52) -9.464 ( 1.129) B 18.04 (3.77) 29.82 (6.11) -11.782 (1.498) C 14.66 (3.28) 20.75 (4.94) -6.083 (1.712)

A 20.78 (1.72) 19.21 (1.70) 1.571 (0.459) B 20.82 (2.14) 19.52 (1.83) 1.304 (0.588) C 17.91 (1.72) 17.33 (2.01) 0.583 (0.766)

Bicarbonate (mmoVL)

Base excess (mmoVL) A -4.53 (1.79) -3.78 (1.64) -0.750 (0.459) B -5.043 (1.58) 4.130 (1.65) -0.913 (0.477) C -5.666 (1.77) -5.166 (2.03) -0,500 (0.780)

DISCUSSION

The complex mechanism of lipid peroxidation, includ- ing the formation of malondialdehyde, is known to require the participation of highly reactive superoxide (02J and singlet oxygen ('OJ in the chain of biochem- ical reactions. Lipid peroxidation occurs in any part of the body where these free radicals are present5."-13. In investigating these chemical reactions at birth, the lev- els of the major product of lipid peroxidation, malondi- aldehyde, were determined in umbilical arterial and venous blood.

Currently, determination of umbilical arterial acid- base status provides the only widely accepted assess- ment of perinatal asphyxia, but pH and base excess reflect only fetal metabolic adjustments and therefore are not directly related to fetal damage, except where extreme values exist14J5.

The findings of Rogers et ~ 1 . ' ~ also demonstrate cellu- lar damage by free radical activity following hypoxia and reperfusion during labour, and support the use of lipid peroxidation as a marker of fetal hypoxia in prefer- ence to acid-base balance16. Wang et al. l7 demonstrated the association between lipid peroxide production and

Table 2. Spearman's rank correlation analysis of umbilical arterial levels of malondialdehyde and acid-base status and duration of labour. Values are given as correlation coefficients (P values). A = cephalic control group; B = nonacidaemic breech group; C = acidaemic breech group.

PH PCO, PO, Bicarbonate Base excess Duration of labour

A (n = 28) -0.732 0.620 -0.303 -0.342 -0.575 0.026 (0.001) (0.001) (0.098) (0.628) (0.001) (0.156)

(0.001) (0.01) (0.01) (0.01) (0.072) (0.521)

(0.001) (0.01) (0.01) (0.05) (0.01) (0.44 1)

B (n = 23) -0.834 0.599 -0.589 -0.575 -0.161 0.126

c (n = 12) 4.792 0.7 15 -0.774 -0.657 -0.796 0.189

B+C (n = 35) -0.8 13 0.777 -0.7 15 -0.702 -0.4 1 3 0.213 (0.001) (0.01) (0.01) (0.01) (0.05) (0.220)

0 RCOG 2000 Br J Obstet Gynaecol 107,982-986

LIPID PEROXIDATION I N UMBILICAL ARTERIAL BLOOD 985

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9 -

8 -

7 -

8 -

2 5 -

$ 4 -

3 - 2 -

1 -

0,

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Table 3. Umbilical arterial malondialdehyde (MDA) levels at birth in the groups. Values are given as mean (SD).

-

A 0

A A 0 0

mo 0 0

AA 0 0.0 0 o n 0

A A Y A -0 0 0

Y A A 0

Y A A A A o o n A A b

Cephalic presentation Nonacidaemic breech Acidaemic breech Total breech (n = 28) (n = 23) (n = 12) (n = 35)

7.3 - 7,25 -

7,2 -

7.15 -

MDA (pmoyL) 1.513 (0.12) 1.743 (O.l)* 1.92 (0.16)' 1.8 (0.15)*

*P < 0.0001 vs control. 'P = 0.0025 vs Group B.

acid-base balance at delivery, where it was found that umbilical arterial malondialdehyde and its arterio- venous difference were negatively associated with pH and PO,, and positively associated with pCO,, They sug- gested that lipid peroxide measurements may be a more sensitive indicator of intrapartum fetal stress than acid-base balance".

The values for umbilical arterial-venous cord blood acid-base status and malondialdehyde levels in 35 breech newborns and 28 cephalic newborns (control) are reported in our study. The significant difference in arterial-venous levels of p0, and pC0, suggested good placental function in all the deliveries. We found a sig- nificant negative correlation between malondialdehyde levels and pH, pOz, bicarbonate, base excess and a posi- tive correlation between malondialdehyde and pC0, (Table 2). In addition, significantly higher umbilical arterial malondialdehyde levels were found in the aci- daemic breech group than in the nonacidaemic breech group (P = 0.0025) (Table 3). The umbilical arterial malondialdehyde levels in the nonacidaemic breech group and total breech group were also higher than the controls (P c 0-OOOl) (Table 3).

One important result of our study is that umbilical arterial malondialdehyde concentrations are increased even in the breech infants without acidaemia, compared with the controls. This suggests that damage due to free radicals may be because of breech delivery itself; but this hypothesis should be tested in a future study.

7,4

7,35 A

Y O

7,i 4 1 1.5 2 2.5

Malondialdehyde (nmol/ml)

Fig. 1. Relationship between umbilical arterial pH and malondialdehyde concentration according to cephalic and breech deliveries. A = cephalic; 0 = breech.

Acknowledgement The authors would like to thank Dr M. Meyer of the Institute of Medical Statistics and Documentation, Friedrich-Alexander-University, Erlangen-Niirnberg, Germany, for his expert statistical guidance.

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Accepted 15 January 2000

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