Indian Pediatrics 1999; 36:659-668

Saroj Kumar Singh, Tarun Dua, Anita Tandon, Sudarshan Kumari, Gibanananda Ray* and Sanjay Batra*

From the Departments of Pediatrics and Biochemistry*, Kalawati Saran Children Hospital, New Delhii-11O 001, India.

Reprint requests: Dr. Saroj Kumar Singh, C-3, 4 Rajpur Road, Tis Hazari, Delhi - 11O 054, India.

Manuscript received May 26, 1998; Initial review completed July 27, 1998;
 Revision Accepted December 2, 1998.

Objective: To compare the activities of key antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase] and the level of malonyl dialdehyde (MDA) in neonates with hypoxic ischemic encephalopathy (HIE) and controls. Design: Prospective cross sectional study. Setting: Tertiary care level 11 neonatal unit of teaching hospital. Methods: Fifteen term new borns with HIE were registered for the study whereas normal term appropriate for gestational age babies were enrolled as controls. Clinical features were recorded. Activities of SOD, GPx, catalase and the content of MDA were estimated at 24 hours of age. The results obtained were statistically analyzed. Results: Activities of SOD and Catalase and the level of MDA were significantly higher in cases as compared to the controls. There was however no significant alternation in the activity of GPx levels in both the groups. Conclusion: The increased activities of antioxidant enzymes are unable to counteract the excessively generated oxidants in vivo, as is evident from the increased MDA levels. Hence, oxygen free radicals may playa significant role in the pathogenesis of HIE.

Key words: Catalase, Gluatathione Peroxidase, Hypoxic ischemic encephalopathy, Malonyl dialdehyde, Superoxide dismutase.

FREE radicals are generated during . stepwise reduction of molecular oxygen and are highly damaging. They pose serious threat to vital organs, tissues, polyunsaturated fatty acids (PUF As) of cell membranes and nucleic acids of cells(1-4). PUFAs are most susceptible to injury and this self perpetuating destruction of PUFAs is known as lipid peroxidation(4). Superoxide dismutase (SOD), gluatathione peroxidase (GPx) and catalase are three important antioxidant enzymes which are found in all aerobic and aerotolerant anaerobic organisms protecting biological structures from free radical mediated injury(5,6). In experimental animals brain injury is reported to be caused by superoxide radical (Ozl and hydrogen peroxide (HzOJ(7). Perinatal asphyxia is associated with intra partum or postpartum hypoxia/ischemia followed by reventilation. The role of free oxygen radicals is implicated in animals and newborns(8,9). Hypoxic ischemic insult followed by resuscitation leads, not only to primary cellular injury but to a delayed secondary injury 24-48 hours later(10). The present study was conducted to elucidate the alternations in the expression of SOD, GPx and catalase and the levels of MDA as an index of lipid peroxidation (LPO) during hypoxic ischemic encephalopathy (HIE) in comparison to control neonates.

Subjects and Methods

Neonates with birth asphyxia and HIE born in our hospital over a period of five months from January 1996 to May 1996 constituted the study material. Fifteen term inborn neonates (gestational age 37-41 weeks) with birth asphyxia (Apgar score 4 or less at 1 minute of life) and HIE surviving for 24 hours or more were included in the study. Nineteen normal term new borns (selected at random) served as controls. In all neonates satisfying selection criteria, detailed baseline information regarding maternal problems and neonatal variables was recorded. Gestational age was assessed by menstrual period and by the method of Ballard(II). Staging of HIE was done according to the criteria of Sarnat and S am at (12). Final enrollment of the cases was done at 24 hours of age and samples were collected.

Blood samples were collected at 24 hours age in heparinized vials for estimation of the activities of SOD, GPx, and catalase and the level of MDA. The activity of SOD was determined by following the inhibition of the auto-oxidation of L-adrenaline under specified conditions(l3). Activity of GPx was monitored at 340 nm by the method of Lepold and Wolfgang(l4), whereas activity of Catalase was assayed by determining the de- composition of HP2 at 230 nm(l5). Lipid peroxidation in the serum was measured in the terms of MDA by the thiobarbituric acid reaction(l6). No information about the sample type was provided to the biochemist carrying out the investigations.

Data was processed and analysed on IBM compatible PL/XT using EPI-Info program. Differences in distribution were anaysed by Students "t" test (ANOV A) or otherwise with the Kruskal Wallis test. Differences in pro- portions were analyzed by using Chi-Square test or by the Fisher exact test if sample size was small.

Results

Of the 15 cases Qf HIE enrolled for the study, three cases were in stage 1, four cases in stage 2, while eight cases were in stage 3. All cases were put on oxygen just after delivery. Respiratory distress in two cases was due to meconium aspiration syndrome and secondary to HIE in six cases. Nineteen normal term babies who served as controls had uneventful stay in the hospital. The clinical profile of cases and controls' is described in Table /.

TABLE 1

 Comparison of Clinical Profile

Variables	Controls (n=19)	Study group (n=15)
Birth Weight (g)
Mean (range)	2810(2520-3540)	2470 (2250-2710)
Gestational Age (wks)
Mean (range)	38.89 (37-41)	38.33 (37-41)
Vaginal: Cesarean birth	17:2	12:3
Maternal problem (%)	Nil	9(60)
Neonatal Morbidities
Intracranial Hemorrhage (%)	-	1 (6.7)
Meningitis (%)	-	1 (6.7)
Respiratory distress (%)	-	8(54)
Late onset septicemia ((%))	-	10 (67)
Metabolic problems (%)	-	5 (33)
Hyperbilirubinemia (%)	-	4 (27)
Duration of administration of oxygen (h)
Mean (range)	-	71.33 (24-350)
Oxygen concentration (FiO2)	-	0.4-0.8 (Median 0.6)
Neonatal Mortality (%)	-	5 (33)

Table II compares the values of SOD, GPx, and catalase and MDA in cases and controls. The activities of SOD and catalase were significantly raised in HIE patients as compared to the control group whereas no significant difference in the expression of the GPx was observed in HIE cases and controls. MDA was significantly higher in cases as compared to the controls.
 

TABLE II

Comparison of Levels of SOD, GPx, Catalase and MDA

Parameters	Cases (n=15)	Controls (n=19)
SOD (units/ml)	12.4±4.1**	2.24±1.3
GPx (n mol/min/dl)	4.1±2.0	3.1±1.1
Catalase (µmol/min/dl)	14.72±4.3*	4.8±2.8
MDA (µmol/h/dl)	21.8±8.4**	2.27±1.17

The activities of SOD, GPx and catalase and level of MDA in various stages of HIE are given in Table III. Activities of SOD and catalase and level of MDA were significantly higher in all three stages of HIE as compared to controls while activity of GPx was raised only in HIE stage I.

TABLE III

Values of SOD GPx and Catalase in Various Stages of HIE

HIE stage	SOD units/ml	GPx (n mol/min/dl)	CAT (µmol/min/dl)	MDA (µmol/h/dl)
I (n=3)	7.55±1.06*	6.59±0.26*	8.67±0.48*	11.08±1.7*
II (n=4)	9.33±0.76*	3.64±1.60	11.17±1.06*	14.51±1.11*
III (n=8)	16.0±1.06*	3.46±1.48	17.25±2.42*	28.73±1.75

Discussion

Fluxes of O2- generated enzymatically or photochemically lead to damage to variety of cellular structures either directly or by involving a cooperative interaction with HP2 which results in the production of violently reactive oxidants possibly hydroxyl radical (.OH- or singlet oxygen(l7). Superoxide radicals are generated by brain during asphyxia and reventilation in new born pigs(l8). The exact mechanism of superoxide generation is not

known with certainty. Various stimuli for superoxide production are sudden rise in arterial pressure at onset of reventilation, re- introduction of oxygen to the ischemic brain, excessive release of excitatory aminoacid transmitters or production of superoxide anion via endoperoxide synthase pathway during reventilation and reintroduction of oxygen to brain (18-22). The most commonly implicated mechanism for generation of oxygen free radicals in asphyxia is hypoxanthine/xanthine oxidase system. Hypoxanthine accumulates in the tissues, plasma and other body fluids during hypoxia. When high concentration of oxygen is administered to hypoxic patient during reperfusion/resuscitation, large amount of oxygen free radicals are produced(23-25). In hypoxia very high levels of hypoxanthine (close to 1 mmol) were measured in cerebrospinal fluid and after intrauterine hypoxia, washing out of hypoxanthine is more pronounced as compared to normal new borns(26,27). In cats after experimental brain injury, superoxide radical and H₂0₂ were responsible for vasodilation of cerebral arterioles and hydroxyl radical produced in the presence of free iron in cerebrospinal fluid led to vascular injury(7). A similar mechanism may be responsible for vascular injury in HIE.

In our series the level of SOD and catalase were significantly high. This may be due to production of .0₂^- and H₂0₂ in HIE cases and subsequent upregulation of antioxidant enzymes SOD and catalase. There was no significant difference in level of GPx in HIE cases as compared to controls except in HIE stage I. The increased level of MDA indicates that the upregulation of SOD and catalase was not able to prevent lipid peroxidation by oxygen free radicals. Recent reports on changes in free radicals in both asphyxia and hypoxic ischemic encephalopathy have shown increased level of SOD, .02-, H₂0₂, xanthine oxidase and lipid peroxidation in cerebrospinal fluid of asphyxiated cases as compared to controls(8,9).

Oxidative stress occurs if balance I between cellular antioxidant defences (SOD, GPx, catalase) is disturbed. According to various studies SOD and catalase are indirect evidence of free radical mediated injury(28). More subtle cooperation involves protection of SOD by catalase and peroxidase against inactivation by H₂O₂. On the other hand SOD protects catalase and peroxidase against inactivation by .0^-2, GPx in a reduced state is susceptible to inactivation by H₂O₂ which is very rapid initially but does not proceed to completion. This may be the reason why GPx level did not increase in HIE(29). In a recent study on asphyxiated neonates, level of LPO' was maximum in the mortality group followed by the morbidity group and controls. The activity of SOD was directly correlated and activity of GPx and level of LPO were inversely related to outcome in asphyxiated neonates(30). It was presumed to be due to inactivation of SOD and upregulation of GPx but these enzymes were not able to prevent LPO completely hence asphyxia related injury was documented.

On the basis of our study it is difficult to say whether free radicals mediated injury might have occurred during asphyxia or reventilation. Most of the studies have demonstrated that oxygen free radicals are generated during reperfusion/reventilation stage and free radical scavangers and calcium antagonists have beneficial effect(31 ,32). It is thus concluded that free radical mediated injury as evidenced by raised MDA level plays a significant role in pathophysiology of birth asphyxia and HIE. It may lead to upregulation of antioxidant enzymes but the increased activities of these enzymes were unable to scavange the free radicals. Due to lack of normative age dependant data of level of antioxidant enzymes, these enzymes, cannot be used to assess outcome. Oxygen should be administered to the cases of HIE only if it is needed to maintain normal blood gas levels. . Liberal use of oxygen may in- crease asphyxia related injury by producing oxygen free radicals. Antioxidants may playa crucial role in management but this hypo- thesis needs more studies with larger sample sizes.