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It has already been more than a quarter century since the first description of CLD in premature infants, but the overall incidence of CLD has changed little in association with the decline of RDS mortality in extremely low birth weight infants due to great progress in neonatal intensive care(1). The implication of oxygen toxicity in a primary etiologic factor of CLD has been advanced repeatedly(2), but little “in vivo” evidence has been obtained(3).

It is widely accepted that uric acid is one of the most effective water-soluble antioxidants in human plasma and that its plasma concentration is markedly higher than those found in most other mammals as a consequence of the loss of uricase activity, which further metabolizes uric acid to allantoin. During the perinatal period, the concentration of uric acid in the cord blood has generally been found to be somewhat higher than simultaneous maternal levels, and to further increase during the first 24 h of life(4). Therefore, especially in newborn infants, it is possible that uric acid may play a key role in the protection against oxidative stress. Ames et al.(5) have proposed that during the course of human evolution and accompanying the loss of the synthesis of ascorbic acid, uric acid may have taken over some of the antioxidant functions of ascorbic acid. When uric acid acts as a sacrificial antioxidant, it is oxidized to yield various oxidation products among which allantoin is the most stable and most abundant(6). It has therefore been proposed that allantoin might be a “marker” of free radical generation in vivo(7). Indeed, we have recently reported the elevation of plasma allantoin levels in untreated patients with Wilson's disease(8).

In the present study, we investigated the possible involvement of oxygen radicals in CLD by quantitating the plasma allantoin concentration as anin vivo marker of free radical generation.

METHODS

Subjects. Nineteen infants admitted to the Neonatal Intensive Care Unit of Osaka Medical College between August 1992 and January 1995 were entered into this study. Infants with congenital heart disease, multiple malformations, and culture-proven sepsis were excluded. These infants were divided into two groups: 10 infants who subsequently developed CLD and 9 without CLD. The clinical characteristics of the infants are summarized inTable 1. All of them had respiratory distress severe enough to require mechanical ventilation at birth, were diagnosed as having RDS, and received surfactant replacement therapy with a single dose of surfactant TA (Tokyo Tanabe Co., Tokyo, Japan) at an average of 1.5 h of life(range: 0.5-2.0 h). The diagnosis of RDS was made on the basis of a combination of the characteristic clinical features and radiographic criteria with negative bacterial cultures, and was confirmed by a stable microbubble test of gastric aspirates. Thereafter, they were mechanically ventilated with a frequency of 10-40 breaths/min, a peak pressure of 15-20 cm H2O, and a positive end-expiratory pressure of 3-5 cm H2O to maintain arterial oxygen tension between 60 and 80 mm Hg for at least the first 5 d of life. CLD was defined as the requirement for extra oxygen supplementation beyond 28 d of age associated with symptoms of persistent respiratory distress and hazy lung fields on chest x-ray films(9). None of the infants had a plasma IgM level >20 mg/dL or a C-reactive protein level >0.25 mg/dL on the first day of life. We obtained heparinized blood samples within the first 24-48 h after birth by salvaging a remnant left after the analysis of arterial blood gases. All infants received i.v. glucose infusion without enteral feeding until the time of investigation. The study protocol was approved by the ethical committee of our college hospital, and the studies were performed after informed consent was obtained from the parents.

Table 1 Clinical characteristics of study neonates
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Determination of allantoin. The plasma allantoin level was measured using a slight modification of the method described in our previous report(8). The changes were as follows. Separated plasma was ultrafiltered though a Millipore Ultrafree C-3LTK filter (Nihon Millipore Ltd., Tokyo, Japan) at 5000 × g for 30 min, and a 20-μL aliquot of the filtrate was injected directly into an anion-exchange column(Irika SAX-1010, 4 × 250 mm; Irica Instruments Inc., Kyoto, Japan) with a mobile phase of 125 mM NaCl containing 1 mM K2HPO4 adjusted to pH 6.75 with HCl and a flow rate of 0.7 mL/min. The HPLC system was identical to that used in our previous study(8). A fraction covering the retention time range of 5.5-10 min, where allantoin was known to be eluted, was collected and then evaporated to dryness at 40°C under vacuum. The residue was reconstituted in 80 μL of 0.5 M NaOH, heated in a boiling water bath for 20 min, and then treated with 10 μL of 5 M HCl and finally with 10 μL of a 3 mM solution of 2,4-dinitrophenylhydrazine in 1 M HCl while being heated for 10 min. Detection of glyoxylate 2,4-dinitrophenylhydrazone, a derivative of allantoin, was performed as described previously(8). The recovery of three different concentrations of added allantoin (10, 20, and 50 μM) to plasma averaged 96.7% in five replicate analyses of each plasma sample.

Determination of uric acid. Plasma uric acid was measured by HPLC according to the method of our previous study(8).

Evaluation of total extra oxygen supplementation and mechanical ventilation. The total extra oxygen supplementation provided and the number of mechanical ventilations within the first 48 h after birth were determined by the following equations;

  • total extra oxygen supplementation = supplemented extra oxygen concentration [%] (fraction of inspired oxygen - 21) × duration [h].

  • total mechanical ventilations = intermittent mandatory ventilation rate setting [times/min] × duration [min].

Statistical analysis. The statistical significance of differences between groups was evaluated with nonparametric Mann-WhitneyU test. Values of p < 0.05 were considered to be statistically significant.

RESULTS

As shown in the Table 1, there were no differences between the infants with and without CLD regarding gestational age, birth weight, Apgar score at 1 min, sex distribution, and the incidence of cesarean section. There was also no significant difference in the total number of mechanical ventilations within 48 h after birth. However, the total extra oxygen supplementation within 48 h after birth was significantly higher in the CLD group.

The plasma allantoin concentration within 24-48 h after birth was markedly elevated in the CLD infants (mean ± SD, 25.9 ± 9.8 μM), being more than double the level in the non-CLD infants (11.0 ± 5.7 μM), as shown in the Figure 1. The allantoin/urate ratio was also significantly higher in the CLD group (5.8 ± 2.0% for CLD infants and 2.4 ± 0.9% for non-CLD infants), reflecting the similar levels of uric acid in the two groups (448.9 ± 114.7 μM for CLD infants and 472.5 ± 176.5 μM for non-CLD infants).

Figure 1
figure 1

The concentration of allantoin and the ratio of allantoin to uric acid in plasma of premature infants with or without CLD within the first 24-48 h after birth. Closed circles represent the allantoin concentration and open circles indicate the allantoin/urate ratio. Closed diamonds on the vertical bars indicate the mean ± SD.

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DISCUSSION

Preterm infants are thought to be hypersusceptible to oxidative stress because of inadequate antioxidant protection, due to low level of plasma radical scavengers such as α-tocopherol(10, 11), marked deficiency of metal binding proteins such as transferrin(12) and ceruloplasmin(13) for the protection of metal-catalyzed free radical reactions, and low activities of antioxidant enzymes such as catalase(14) and glutathione peroxidase(15). Hyperoxic exposure itself, although essential for survival of the RDS infants, probably induces an excessive production of reactive oxygen metabolites in the respiratory system.

In the present study, the significantly higher indices of total extra oxygen supplementation within 48 h after birth in the CLD infants suggested a role for oxygen radicals in the pathogenesis of CLD. Indeed, the significant elevation of the plasma allantoin level and the allantoin/urate ratio in the CLD infants provided clear evidence of increased generation of oxygen radicals in the plasma aqueous phase in the early neonatal period when compared with non-CLD infants. Many investigators have proposed that hyperoxia is one of the principal factors in the pathogenesis of CLD(3, 16). In contrast, Varisla et al.(17) studied ethane and pentane in expired air and reported that the degree of prematurity was the single most important factor explaining free radical-mediated lipid peroxidation and that hyperoxia failed to show a significant association with level of lipid peroxidation in premature infants. A possible explanation for this discrepancy is that allantoin is a marker of free radical generation in the aqueous phase of plasma and not a marker of free radical-induced tissue damage, whereas volatile products of lipid peroxidation such as ethane and pentane are indicators of tissue damage caused by free radicals rather than the extent of free radical generation.

In conclusion, our results suggest a role for oxygen radicals in triggering CLD. In addition, our data also suggest that the plasma allantoin concentration and the allantoin/urate ratio may be useful early predictors of the development of CLD. As far as we are aware, studies on the plasma allantoin concentration in CLD infants have not previously been reported. Further studies in a larger number of patients are necessary to confirm our hypothesis and such an investigation is currently in progress at our laboratory.