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Perinatal hypoxia is known to be one of the major causes of perinatal morbidity and mortality. Each kind of oxygen deficiency is combined with accelerated purine degradation which could lead to increased formation of superoxide radicals and H2O2 via xanthine oxidase reactions. It is unknown if there is really increased generation of oxygen free radicals and increased rates of lipid peroxidation processes during and after perinatal hypoxia. The cascade of peroxidation reactions of unsaturated fatty acids leads to the formation of aldehydes as secondary lipid peroxidation products. Those aldehydes can act as “second toxic messengers” of free radicals(1–3). MDA is the aldehydic lipid peroxidation product, which was studied most intensively. Even MDA is cytotoxic and chemically reactive. Some of the aldehydic lipid peroxidation products, such as the 4-hydroxyalkenals, are even more aggressive than MDA and lead to cell damage at nanomolar concentrations. The most important representative of hydroxyalkenals is HNE which is generated from ω-6 polyunsaturated fatty acids. HNE has high cytotoxic and genotoxic activity by reactions with proteins and inhibition of energy-requiring processes(4–6).

The aim of this study is to assess lipid peroxidation after perinatal hypoxia by measurements of MDA and HNE levels and glutathione status in umbilical cord blood of newborns. Measurements of hypoxanthine and uric acid indicate the rate of purine nucleotide degradation which is accelerated during oxygen deficiency.

METHODS

Patients. Samples of 109 newborns from the Department of Neonatology, Medical Faculty (Charité) of Humboldt University, Berlin, Germany, were measured. Some clinical data characterizing the health status of the patients are shown in Table 1. The patients were distributed into five groups considering the gestational age, clinical signs of asphyxia in connection with the necessity of reanimation procedures, and pH of the umbilical artery.

Table 1 Clinical data of patients
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Groups 1-3 consisted of term babies in contrast to groups 4 and 5 with preterm babies. Group 1 consisted of 49 healthy term newborns serving as the control group. Group 2 was composed of 12 term babies with acidosis (umbilical artery pH was less than 7.20) but without disturbances of adaptation after birth, i.e. with a normal Apgar score 5 min after birth. Group 3 consisted of 14 term newborns suffering from asphyxia and with a decreased Apgar score. The Apgar score assessed 5 min after birth was less than 8 points. All of these babies showed disturbances in the fetal cardiotocogram and needed reanimation and oxygen supplementation longer than 10 min after birth. In the prenatal period two thirds of them had meconium in the amniotic fluid. Group 4 included 20 healthy preterm infants with a gestational age ranging from 34 to 36 wk and a normal Apgar score. Group 5 was composed of 19 preterm infants, with a gestational age from 26 to 36 wk, indicating disturbances in the fetal cardiotocogram and in postnatal adaptation. Reanimation procedures and oxygen therapy longer than 10 min were necessary in all of these cases.

In this study infants of only healthy mothers were included. The mothers did not receive drugs other than betamethasone or ambroxol to induce fetal lung maturity or the usual tocolytic sympathomimetica, e.g. fenoterol, to prevent preterm delivery in the group 5.

Procedures. The assay of MDA was performed in a very specific manner by means of the reaction of MDA in the presence of thiobarbituric acid and after HPLC separation of a MDA-thiobarbituric acid conjugate according to Wong et al.(7). The methodologic advances of this method for MDA measuring were described by Grune et al.(8).

HNE was determined as described by Sommerburg et al.(9). The method includes the carbonyl derivatization with 2,4-dinitrophenylhydrazine, the thin-layer chromatographic separation into three zones of 2,4-dinitrophenylhydrazine carbonyls, and the final HPLC analysis of dinitrophenylhydrazone of HNE which was detected at 378 nm. The interassay coefficient of variation was 0.22, and the within-assay coefficient of variation was 0.05.

GSH was measured spectrophotometrically as a product of Ellman's reaction at 412 nm as described by Beutler et al.(10). GSSG was determined fluorimetrically after its reaction witho- phthaldialdehyde according to Hissin et al.(11) at 350 nm (excitation) and 420 nm (emission).

Hypoxanthine and uric acid were determined by means of capillary zone electrophoresis as described previously(12). Separations were performed in an uncoated silica capillary on a SpectraPhoresis 1000 system with UV detection over the range 200-300 nm.

GSH and GSSG were measured in red blood cells, and MDA, HNE, hypoxanthine, and uric acid were measured in the plasma of blood samples taken from the placental part of the umbilical cord. For measurements, samples were used which became available only within 15 min after the babies' birth. To prevent coagulation, heparin was added. The samples were centrifuged directly after sampling and stored in liquid nitrogen. Plasma and red blood cells were stored in liquid nitrogen not longer than 3 d before analyses.

For statistical analyses the parameter-free Wilcoxon test were used.

RESULTS

Aldehydic products of lipid peroxidation MDA and HNE, reduced and oxidized glutathione, and the purine compounds hypoxanthine and uric acid were measured in umbilical arterial cord blood of 109 newborns. In dependence on increased gestational age, increased concentrations of MDA and HNE as well as reduced glutathione were found, whereas there were no significant changes of oxidized glutathione and purine compounds with gestational age(Fig. 1–3). The GSSG/total glutathione ratio decreased with gestational age of the newborns (Fig. 2C). This ratio was calculated on the basis of sulfhydryl units as [2GSSG:(GSH + 2GSSG)]. It was assessed to be about 3.2% in infants with a gestational age of 34 wk and decreased to about 2.1% in newborns with a gestational age of 41 wk(Fig. 2C).

Figure 1
figure 1

Concentrations of HNE (A) and MDA(B) in umbilical cord plasma of healthy newborns with dependence on gestational age. Total number of newborns was 69. The mean values are represented as a short line. The correlation coefficient of HNE concentrations amounted to 0.79 and the correlation coefficient of the MDA level to 0.68.

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Figure 3
figure 3

Concentrations of hypoxanthine (A) and uric acid (B) in umbilical cord plasma of healthy newborns with dependence on gestational age. Total number of newborns was 61. The mean values are represented as a short line.

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Figure 2
figure 2

Level of GSH (A) and GSSG (B) and the ratio [2GSSG:(GSH + 2GSSG)] (C) in red blood cells of umbilical cord blood of healthy neonates with dependence on gestational age. Total number of newborns was 69. The mean values are represented as a short line. The correlation coefficient of GSH concentrations amounted to 0.70.

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In the Figures 4 to 6, groups 1-5 of the newborns which were characterized above were compared. InFigure 4 the MDA and HNE levels in neonates of the different groups are documented. The MDA level amounted to 1.8 nmol/mL serum in healthy term newborns and increased to about 2-fold in term newborns with acidosis and asphyxia (p < 0.001 in comparison with the control group of healthy term newborns). In preterm neonates a more than 5-fold increase of MDA values from 0.6 nmol/mL in healthy preterm babies to 3.3 nmol/mL in preterm asphyctic infants was noted with a high significance, too(p < 0.001). HNE levels in umbilical cord plasma of full-term healthy neonates amounted to about 0.3 nmol/mL serum. After perinatal complications, the HNE concentrations increased significantly in the group of term newborns with acidosis to about 0.5 nmol/mL and in term neonates suffering from asphyxia to about 0.6 nmol/mL (p < 0.001 in comparison with the control group of healthy term newborns). In healthy preterm neonates with a gestational age from 34 to 36 wk the mean HNE level was 0.1 nmol/mL. HNE increased up to 0.4 nmol/mL in preterm newborns after perinatal asphyxia (p < 0.001).

Figure 4
figure 4

Concentrations of HNE (A) and MDA(B) in umbilical cord plasma of healthy (group 1), acidotic (group 2) and asphyctic (group 3) term newborns and of healthy (group 4) and asphyctic (group 5) preterm neonates. Values are given as mean values ± SEM with number of estimations in parentheses. Statistical differences are represented as **(p < 0.01), ****(p < 0.001) in comparison to group 1 values and as + + + +(p < 0.001) in comparison with group 4 values.

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Figure 6
figure 6

Concentrations of hypoxanthine (A) and uric acid (B) in umbilical cord plasma of healthy (group 1), acidotic(group 2), and asphyctic (group 3) term newborns and of healthy (group 4) and asphyctic (group 5) preterm neonates. Values are given as mean values ± SEM with the number of estimations in parentheses. Statistical differences are represented as *p < 0.05 in comparison with the group 1 values and as +p < 0.05 and ++p < 0.01 in comparison with group 4 values.

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In Figure 5 concentrations of GSH, GSSG, and of the GSSG/total glutathione ratio in newborns of the five different groups are shown. In term newborns the erythrocytic GSH level amounted to 2.18 μmol/mL of red blood cells for healthy infants without acidosis or asphyxia. GSH diminished to 1.13 or 1.24 μmol/mL of cells (p < 0.001) in term newborns suffering from acidosis or asphyxia (Fig. 5A). The GSSG level stayed nearly constant with about 21-24 nmol/mL of red blood cells for all groups of term infants (Fig. 5B). In preterm infants GSH values declined from 1.13 (for healthy, nonasphyctic preterm newborns) to 0.63 μmol/mL of cells for preterm newborns suffering from asphyxia and characterized by a decreased Apgar score (p < 0.001). The GSSG level in preterm newborns increased after perinatal asphyxia from 22.8 to 47.9 nmol/mL of cells (p < 0.05). The ratio[2GSSG:(GSH + 2GSSG)] amounted to 2.40% in healthy term newborns. In acidotic or asphyctic term infants this ratio increased to 4.04 (p < 0.005) or 3.54% (p < 0.05 compared with the control group of term infants). In the group of healthy premature babies this ratio was assessed to about 4% (p < 0.005 in comparison with healthy term infants). After asphyxia there was found a distinct increase in preterm infants to almost 9% (p < 0.05 in comparison with healthy preterm newborns)(Fig. 5C).

Figure 5
figure 5

Concentrations of GSH (A), GSSG(B), and the ratio of [2GSSG: (GSH + 2GSSG)] (C) in red blood cells of umbilical cord blood of healthy (group 1), acidotic (group 2), and asphyctic (group 3) term newborns and of healthy (group 4) and asphyctic(group 5) preterm neonates. Values are given as the mean values ± SEM with the number of estimations in parentheses. Statistical differences are represented as *p < 0.05, ***p < 0.005, and ****p < 0.001 in comparison with group 1 values and as +p < 0.05, + + + +p < 0.001 in comparison with group 4 values.

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Figure 6 shows the level of hypoxanthine and uric acid in the various groups of term and preterm neonates. The mean concentration of hypoxanthine measured in term infants with asphyxia was higher as compared with healthy term infants but without statistical significance (13.4 nmol/mL in comparison with 11.0 nmol/mL). In preterm newborns with asphyxia a significant increase of hypoxanthine levels was found in comparison with hypoxanthine values in healthy preterm newborns (15.3 nmol/mL in comparison with 9.6 nmol/mL) (p < 0.01). Uric acid amounted to 307 nmol/mL in the control group (healthy term newborns) and rose by 20 or 30% in term newborns with acidosis or asphyxia (p < 0.05). In the groups of preterm neonates uric acid levels increased even from 305 nmol/mL (healthy preterm newborns) to 481 nmol/mL, i.e. by about 60%, in preterm newborns with asphyxia (p < 0.05).

DISCUSSION

The formation of HNE in the course of lipid peroxidation was proved in well known biologic model systems for oxidative stress (e.g. NADPH oxidation by liver microsomes, CCl4 intoxication) and is useful for measurements of oxidative loading(4). Until now nothing has been reported about HNE levels in newborns. In this study HNE concentrations were measured in umbilical cord plasma of healthy, acidotic, and asphyctic term and of healthy and asphyctic preterm infants.

In full-term neonates the HNE values amounted to about 0.3 nmol/mL and there was a distinct increase of HNE concentrations during the last weeks of pregnancy (from 34 to 41 wk). The MDA levels in umbilical cord plasma showed a clear increase, also depending on gestational age. This accumulation of aldehydic products of lipid peroxidation might be explained by increased pool of polyunsaturated fatty acids as substrates for lipid peroxidation in term infants(13).

The increased GSH level found in term infants in comparison with premature infants was in accordance with findings of other authors(14). The increased concentrations of GSH easily can be explained by higher enzyme activities of the antioxidative defense system, including glutathione reductase and glutathione synthetase at the end of pregnancy(15–17).

After perinatal acidosis or asphyxia, distinctly increased concentrations of HNE and MDA were found, indicating an elevated rate of peroxidation reactions under these circumstances. For evaluation of changes by acidosis/asphyxia the comparison between the following groups of our newborn patients makes sense: groups 2 and 3 with group 1 or group 5 compared with group 4. The raised levels of both MDA and HNE were accompanied by a strong decrease of reduced glutathione values. That could be due to the rapid consumption of GSH via a glutathione peroxidase reaction but additionally to the high reactivity of HNE with sulfhydryl groups.

It is known that oxygen deficiency is accompanied by a rapid degradation of energy-rich purine nucleotides, by accumulation of hypoxanthine as substrate of xanthine oxidase, and by uric acid accumulation. In this study only a minor increase of hypoxanthine levels but a drastic increase of uric acid levels was found after asphyxia, both in term and in preterm infants, but more distinctly in preterm infants. A sensitive increase of hypoxanthine was reported previously(18, 19), but also a great overlapping of hypoxanthine concentrations in healthy and hypoxic newborns(20, 21). Nevertheless, from increased hypoxanthine levels after oxygen deficiency and directly from drastic uric acid accumulation, one can conclude that the xanthine oxidase reaction steps were accelerated(22, 23), which contribute to increased generation of free radicals in asphyctic term and preterm newborns. Free radical generation induces peroxidation of unsaturated fatty acids and, afterward, causes enhanced levels of aldehydic lipid peroxidation products such as HNE and MDA. The concentration of HNE reflects sensitively the level of lipid peroxidation in vivo after perinatal hypoxia and represents a new parameter which is proposed for quantitative evaluation of posthypoxic cellular damage.