Original Article
Indian J Pediatr 1998; 65 : 419-427 i|
Free Oxygen Radicals --Predictors of Neonatal Outcome Following Perinatal Asphyxia Sushma Nangia, Arvind Saili, A.K. Dutta, S. Batra* and G.N. Ray*
Departments of Neonatology and Biochemistry* KaIawati Saran Children's Hospital and Lady Hardinge Medical College, New Delhi A b s t r a c t : The study was undertaken to evaluate the role of free oxygen radicals in asphyxiated
neonates. Thirty term neonates appropriate for gestational age and with severe birth asphyxia (Apgar score of 3 or less at 1 minute of life) formed the study subjects. The levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), creatine phosphokinase (CPK) and lipid peroxidase (LPO) in the CSF of these neonates were estimated between 12 and 48 hrs of life. Enzyme estimation was performed by standard methods and the results were analysed statistically using Multivariate Logistic Regression analysis and non parametric tests namely Kruskal Wallis test and Wilcoxon's rank sum test. Out of the thirty babies, 14 were observed to be neurologically normal, 9 had significant morbidity and 7 died. The SOD levels ranged from 12.4 to 140 units/ml, GPx from 128 to 1933 nmol/min/dl, CPK from 2 to 2098 IU/dl and LPO from 5.4 to 30.8 umol/hr/dl. The SOD and GPx levels had an inverse relationship whereas rise in LPO and CPK levels were directly proportional to the extent of neurological damage and ultimate clinical outcome. CPK levels higher than 140 IU/ml were lethal and associated with 100% mortality whereas all normal neonates had CPK below 37 IU/ml. The levels of antioxidant enzymes can reliably and significantly predict mortality and morbidity whereas level of an enzyme cannot confidently confer normalcy. Hence antioxidant enzyme levels with a cut off value can be a useful marker and serve as a prognostic indicator in times to come. ( I n d i a n J Pediatr 1998; 65 : 4 1 9 - 4 2 7 )
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Key words : Birth asphyxia; Free oxygen radicals; Antioxidant enzymes.
Birth asphyxia is one of the major causes of perinatal mortality and morbidity especially in developing countries. Perinatal asphyxia is associated with intrapartum or postpartum hypoxia/ ischaemia followed by reventilation and reperfusion during resuscitation. The role of free oxygen radicals has been implicated in such reventilation/reperfusion injuries Reprint requests : Dr. Sushma Nangia, B-l/ 1702, Vasant Kunj, New Delhi - 110 070.
following hypoxia. The pathogenesis of cell death following hypoxic ischaemic injury in the developing brain is complex and incompletely understood. It is clear, however, that the hypoxic ischaemic insult followed by resuscitation leads, not only to primary cellular injury during the period of insult, but to a delayed secondary injury 24-48 hrs later. Neurodevelopmental prognosis is directly related to severity of secondary injury.7.17 The mechanisms of this secondary event
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are poorly u n d e r s t o o d but probably involve cellular injury mediated by excess concentration of excitatory neurotransmitters, free oxygen radical formation and lipid peroxidation which in turn leads to a cascade of events. 17 The present s t u d y was undertaken to evaluate the levels of antioxidant enzymes in CSF of babies with severe birth asphyxia and thus ascertain the role of free oxygen radicals in this clinical condition. MATERIALS A N D METHODS
The study was conducted between January '95 to June '95. The material comprised of all the term appropriate for gestational age neonates who had suffered severe birth asphyxia during this period (Apgar score of 3 or less at I minute of life). The CSF sample was taken after informed consent of the parents for antioxidant enzyme level estimation between 12 and 48 hrs of life. Neonates with maternal risk factors for sepsis or those w h o developed sepsis, intraventricular haemorrhage, intracranial haemorrhage or meningitis were excluded from the study. If a haemorrhagic tap occurred during lumbar puncture, the procedure was abandoned and the neonate excluded from the study. Enzymes estimated were superoxide dismutase (SOD), glutathione peroxidase (GPx), lipid peroxidase (LPO) and creatine phosphokinase (CPK). The activity of SOD was determined by the method of Mishra and Fridovich (1971). 2The reaction mixture contained carbonate buffer (pH 10.2), epinephrine and suitable aliquot of enzyme. One enzyme unit is defined as the amount of protein that inhibits the auto-oxidation of
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epinephrine by 50% under specified conditions. It is measured in units/ml. GPx was assayed at 340 nm; the reaction mixture contained PO~ buffer (pH 7.0), NADPH, reduced glutathione, EDTA, sodium azide, glutathione reductase and tbutyl hydroperoxides and suitable aliquot of enzyme (CSF). LPO malonyldialdehyde levels were estimated after 3 hrs. of incubation of CSF by reacting with thiobarbituric acid. The GPx levels were measuredin nmol/ min/dl and LPO in n m o l / h r / d l . The CPK levels were estimated by Randox Kit of Sigma Laboratories on auto analyser REPLY (OLYMPUS) and measured in IU/ml. The results were analysed using Multivariate Logistic Regression analysis taking the original individual values of the four enzymes as independent variables and the outcome as dependent variable and the P value was computed. The variation in the various enzyme levels in relation to the clinical outcome was analysed using non parametric tests namely Kruskal Walli's test and Wilcoxon's rank sum test. One way analysis of variance was used for each enzyme taking the outcome as expired, morbidity and normal survival. Finally, the sensitivity, specificity; the positive and negative predictive values of all the four enzymes were computed and their association tested through Chi-square test. RESULTS The SOD ievels ranged from 12.4 to 142 units/ml, GPx from 128 to 1993 n m o l / m i n / d l whereas CPK ranged from as low as 2 to 2098 I U / m l and malonyl dialdehyde levels ranged from 5.4 to 30.8 u m o l / h r / d l , in the CSF of the thirty neonates taken up for the study.
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SUSHMA NANGIA E T A L
The m e a n level, along w i t h SD of the whole group for SOD was 47.72 and 34.26, for GPx it was 774.17 a n d 509.63, for LPO 16.74 and 6.83 and for CPK it was 57.5000 and 69.6958. The clinical o u t c o m e of the thirty neonates revealed that 14 h a d minor problems initially and stabilised by 72-96 hours of life and were essentially n o r m a l at the time of discharge. N i n e babies h a d a stormy prolonged hospital stay full of various problems and were not neurologically normal even at the time of discharge, constituting the morbidity group. Seven babies succumbed to their illness, constituting the expired group. Five out of nine h a d HIE stage II which lasted for 6-10 days and at the time of discharge, only one baby h a d n o r m a l neurological examination. Two out of the nine h a d m y o c a r d i a l ischaemia a n d CHF and required treatment for the same for 3 and 4 d a y s respectively. Twolbables d e v e l o p e d metabolic seizures d u e to h y p o c a l c a e m i a a n d h y p o m a g n e s e m i a respectively and both these babies also h a d oliguria and deranged kidney function tests.
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Eight out of the total thirty babies had hypoglycemia and two required treatment with h y d r o c o r t i s o n e after full sugar supplementation to 12 m g / k g / m i n . Ten out of thirty babies h a d severe metabolic acidosis (pH < 7.0) a n d fourteen babies h a d HIE II-III, five babies h a d HIE I a n d one of these p r o g r e s s e d to stage II. Eighteen babies h a d deranged KFTs on D2 which persisted in seven till D6. Eight babies h a d d e r a n g e d LFT. Four babies had deranged coagulation profile. To statistically analyse the variation in the various enzyme levels in relation to the clinical outcome, multiple comparison test a n d a non parametric test n a m e l y Wilcoxon's rank sum test were applied using Microstat and Fox pro software packages taking one e n z y m e at a time and the outcome as : expired (group I), morbidity (group II) and normal survival (group III). The results are as shown in Table 1. When expired were compared with the morbidity group, the differences in the enzyme levels were not significant for LPO, SOD and GPx but highly significant for CPK being < .001. When expired were compared with normal
TABLE1. Clinical Outcome in Relation to Enzyme Levels Group I n=7 Expired SOD GPx LPO CPK
30.47 • 11.8 1001.29 • 712 19.52 • 7.22 433.43 • 737
Group II n=9 Morbidity
Group Ill n=14 Normal survival
45.29 + 28.7 781.56 • 558 17.42 _+5.19 32.5 • 20.8
60.9 • 24.5 695.14 • 412 13.31 • 6.07 23.65 • 19.2
FOOTNOTE (1) : Comparison of Group I & Group I! by Multiple Comparison Test illustrates : SOD and LPO Nonsignificant (p > 0.05); GPx Significant (p < 0.05); CPK Highly significant (p < 0.001). (2) : Comparison of Group I & Group III by Multiple Comparison Test illustrates : SOD Significant (p < 0.01); LPO Significant (p > 0.01 < 0.05); GPx Nonsignificant (p > 0.05); and CPK Highly significant (p < 0.001).
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TABLE2. Clinical Outcome as ~.xpired vs Survived in Relation to Enzymes Levels Group I n=7 Expired SOD GPx LPO CPK
30.47• 1001.28• 19.52• 433.42•
Group II n=23 Survived 53.9• 726.82• 14.4• 25.6•
P value
<.05 >.1 <.05 <.0001
Sig.
Sig. NS Sig. HS
Groups I and II compared by Wilcoxon's rank sum test s u r v i v a l g r o u p , the d i f f e r e n c e s in the enz y m e levels w e r e significant for SOD, LPO and highly significant for CPK, the p value b e i n g <.01 b e t w e e n 0.01 a n d 0.05 a n d < .001 respectively. Table 2 d e p i c t s the clinical o u t c o m e as expired (n = 7) vs s u r v i v e d [morbidity (9) + n o r m a l (14), n = 23] in relation to the enz y m e levels a n d here again the differences were statistically significant for SOD, LPO a n d C P K a n d p v a l u e s b e i n g < .05, < .05 and <. 001 respectively. M u l t i v a r i a t e Logistic Regression a n a l y sis (Table 4) w a s d o n e , taking the f o u r enz y m e s as i n d e p e n d e n t v a r i a b l e s a n d the outcome as d e p e n d e n t variable i.e. n o r m a l (normal n = 14) vs a b n o r m a l o u t c o m e (exp i r e d n = 7 + m o r b i d n = 9 = 16) a n d surv i v e d ( n o r m a l n = 14 + m o r b i d n = 9 = 23) vs expired (n = 7).
The a n a l y s i s r e v e a l e d that for s u r v i v a l vs expired (Table 4a) LPO w a s the only ind e p e n d e n t p r e d i c t o r w i t h an o d d ' s ratio (OR) of 1.4 [95')/o CI (1.06-1.71)]. The other e n z y m e s failed to attain statistical significance at m u l t i v a r i a t e levels t h o u g h CPK, LPO a n d SOD w e r e significant at the u n i v a r i a t e level w i t h p v a l u e s of 0.022, 0.021 and 0.042 respectively. Similar analysis (MLRA) w a s c a r r i e d o u t c o n s i d e r i n g the o u t c o m e as n o r m a l vs a b n o r m a l (Table 4b) which revealed LPO and SOD as the ind e p e n d e n t predictors with o d d ' s ratio (OR) of 1.4 (95% CI 1.02-1.78) a n d 0.96 [95% CI (0.95-1.002)] respectively. The other t w o enz y m e s did not attain statistical significance at m u l t i v a r i a t e level t h o u g h at u n i v a r i a t e level SOD, l_PO a n d CPK all w e r e statistically significant w i t h p v a l u e s of 0.011, 0.002 and 0.012 respectively.
TABLE3. Sensitivity and Specificity of Various Enzymes SOD
GPx
LPO
CPK
Sensitivity Specificity Pos. pred. value
71% 87% 62.5%
57% 52% 26.6%
85% 74% 50%
86% 91.3~ 75%
Neg. pred. value False neg False pos.
91% 28% 13%
80% 43(/0 47%
94.4% 14% 26%
95% 14% 8.6%
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Table 3 d e p i c t s the s e n s i t i v i t y , specificity, positive and negative predictive values a l o n g w i t h t h e false n e g a t i v e s a n d the false TABLE 4. M u l t i v a r i a t e
Logistic
Regression
Analysis Independent variables - SOD, GPx, LPO, CPK Dependent Variable - Outcome
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p o s i t i v e s for the v a r i o u s e n z y m e s . T h e sens i t i v i t y of CPK, L P O a n d S O D w a s h i g h b e i n g 86%, 85% a n d 71% r e s p e c t i v e l y . T h e s p e c i f i c i t y w a s h i g h e s t f o r C P K b e i n g 92~ f o l l o w e d b y S O D a t 87% a n d L P O at 74%. T h e p o s i t i v e p r e d i c t i v e v a l u e for C P K w a s 75%; 62.5% for S O D a n d 50% f o r LPO. Similarly, the negative predictive value w a s 95% for b o t h C P K a n d L P O a n d 91%
TABLE4a. Outcome Survival vs Dead (n = 23) (n = 7) Variable entered - LPO Variable LPO Constant i.e OR 1.37
SE
Wald
df
Sig
.301
.118
6.53
1
.0106
6.664
2.346
8.07
1
.0045
B
OR = Exp (B) 1.37
95% CI 1.06 - 1.71 TABLE4b. Outcome - Normal vs Abnormal (n = 14) (n = 16) Variable entered - LPO Variable LPO Constant i.e. OR 1.34
B
.290 -4.449
SE .114 1.818
Wald 6.42 5.99
df 1 1
Sig .01 i ,:, ,0144-
Exp (B) 1.336
95% CI 1.06 - 1.68 Variable in the Equation B
SE
Wald
df
Sig
SOD
- .041
.022
3.51
1
.0610
.9599
LPO
.302
.137
4.87
1
.0273
t.3530
Constant - 2.520 OR - .96 95% CI-0.92-1.002 OR - 1.35 95% CI - 1.02 - 1.78
2.074
1.48
1
.2244
Variable
B = Regression coefficient SE = Standard error df = Degree of freedom Exp (B) or OR = Exponential of B
OR = Exp (B)
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for SOD. The sensitivity ranged from 57 to 86%, specificity from 52 to 91.3%, positive predictive value from 27 to 75% and the negative predictive value from 80 to 95%. To test the statistical significance of the difference in the validity parameters between enzymes, Chi square test was applied and the results revealed p = 0.55 for sensitivity, 0.003 for specificity, 0.062 for positive predictive value and 0.38 for negative predictive value, being statistically significant for specificity and positive predictive value. When considered individually, the SOD levels were the lowest in the most affected new borns and conversely the CPK, GPx and LPO levels were higher in the babies who had an unfavourable outcome. None of the normal babies had low SOD and high GPx levels together. CPK above 140 IU/ml was associated with 100% mortality and all the normal babies had CPK below 37 IU/ml. DISCUSSION
Perinatal asphyxia is an insult to the fetus or newborn due to lack of oxygen (hypoxia) or lack of perfusion (ischaemia) to various organs. 1Almost every organ of the body is affected by hypoxia leading to multisystem failure but predominant insult is to the CNS with very little scope for repair. The mechanism of cellular injury following hypoxio-ischaemia=reventilationreperfusion is poorly understood but is probably mediated by excess concentration of excitatory neurotransmitters, free radicaV formation and lipid peroxidation which in turn leads to a cascade of events.8.9,~o Oxygen free radicals are highly reactive and can initiate chain reactions which form
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new free radicals. Although the life time of each radical is extremely short, its action may continue by an explosive and proliferative generation of new radicals. Free radicals injure biological membranes by lipid peroxidation. 11 Stable degradation products of such processes e.g., malonyldialdehyde, pentate and ethane may, therefore, be used as markers of peroxidation of polyunsaturated fatty acids.12 Experimental interventions in anaesthetised cats to produce fluid percussion brain injury generates superoxide in the brain. 3 This radical enters the cerebral extra cellular space where it dismutates to hydrogen peroxide. Since CSF contains micromolar concentrations of free iron, superOxide and H202 interact via the iron ~ catalysed Haber Weiss reaction to produce hydoxyl radicaD Hydroxyl free radical generation in animal models of ischaemia/reperfusion have been detected by different group of workers and they have also shown that endogenous antioxidant enzymes including SOD, catalase, GPx and thiospecific antioxidant enzymes protect the neurons against oxidative damage caused by cytotoxic hydroxyl and thiyl free radicals. ~ Protection from free oxygen radicals occurs by intervention at different stages of free radical processes by protective agents which are 'scavengers' like SOD, bilirubin, mannitol, DMSO and uric acid, antioxidants like vitamin E and repair agents and antioxienzymes like GPx and catalase which remove prec'ursors of free radicals and are necessary for quick conversion of hydrogenperoxide or peroxyradicals to molecular oxygen and water. In the present study, the level of SOD which is a protective enzyme and comes into play to scavenge the superoxide radical was reduced in the babies who had an
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SUSHMA NANGIA ETAL
unfavourable outcome, suggesting that either this protective enzyme was utilised to neutralize or scavenge superoxide radical and its levels were in turn lowered in the affected babies, (where free oxygen radicals are formed) or that its substrate induced upregulation or stimulation had not occured either due to some defect in the stimulation and synthesis sequences or a defect at the gene level. 16,18 Whatever be the reason of depletion of this enzyme, it has been proved, beyond doubt, in various animal experiments that supplementation with exogenous SOD as shown by Kontos et al (1986) 12eliminates or at least reduces the extent of damage produced by superoxide when compared with those in which no exogenous enzyme was provided. Similar results have been obtained by Patt A et al 1990. 22 Beckman et al 1991 have shown that SOD and catalase supplementation reduced infarct volume in focal middle cerebral artery stroke model in the rat. 17 The levels of enzyme GPx which comes into play to neutralise the precursors of lipid peroxidation namely the perhydroxyl radical HOO (which is the protonated form of superoxide radical) were increased in babies who had an unfavourable outcome possibly because this repair agent's production is upregulated in response to the generation of the radicals it neutralizes. However, the response of this enzyme was not consistent and more work is needed to definitely establish its role in such free radical mechanisms. The levels of LPO (malonyldialdehyde) were high in babies with a poor outcome denoting that when the precursors of lipid peroxidation namely hydroxyl and perhydroxyl radicals is have not been effectively neutralized by the anti-oxidant defences of the body, the free oxygen radicals
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cause lipid peroxidation. The levels of malonyldialdehyde denote that the extent of lipid peroxidation that occurs, the higher is the amount of free oxygen radicals produced in a particular baby, higher is the attack on lipids of cell membranes and higher is the cell damage reflecting higher levels of LPO thus leading to significant morbidity and mortality.3,19 CPK levels are significantly high in asphyxiated infants as documented in several studies. Fernandez et a! 1987, 21measured CPK at 4 and 10 hrs of life in 33 term asphyxiated neonates and followed them for 16 months and found the levels to be significantly high in those who developed neurological sequelae or died compared to those who did not. They have recommended that high CPK activity can be taken as a sensitive indicator of conspicuous brain damage. The results of Multiple Logistic Regression analysis wherein the four enzymes were seen as independent variables and outcome as dependent vtiriable considering survivors vs expired, revealed LPO as the only independent predictor whereas during the univariate analysis SOD, CPK and LPO were all significant. Similar analysis carried out considering the outcome groups as normal vs abnormal revealed enzymes LPO and SOD as the independent predictors though on univariate analysis, CPK LPO and SOD were all significant. These findings essentially show that the various enzymes are highly interrelated and possibly interdependent, hence, indep e n d e n t effect of different enzyme levels did not reach statistical significance at the multivariate level. The possible reasons for this kind of resuits could be due to a small number of dead babies (n = 7) and an overall small
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sample of 30 babies. This s t u d y is a p r e l i m i n a r y d e s c r i p t i v e s t u d y a n d the results s h o w that the a b o v e m e n t i o n e d e n z y m e levels can reliably a n d significantly predict mortality a n d m o r b i d ity b u t the absolute levels of these e n z y m e s c a n n o t c o n f i d e n t l y p r e d i c t a n o r m a l outc o m e in a p a r t i c u l a r n e o n a t e . H e n c e , the level of these e n z y m e s with a cut off value can be a useful m a r k e r of cell d a m a g e and serve as a p r o g n o s t i c indicator in times to come. H o w e v e r , this is a p r e l i m i n a r y s t u d y a n d r e v e a l s initial t r e n d s of e n z y m e s . Larger n u m b e r of cases n e e d to b e studied taking these p a r a m e t e r s sequentially to arrive at m o r e definitive conclusions.
ACKNOWLEDGEMENT The a u t h o r s g r a t e f u l l y a c k n o w l e d g e the statistical g u i d a n c e given b y Dr. NS Murty, D e p u t y Director, I C M R Institute of Cytology & P r e v e n t i v e Oncology, M a u l a n a A z a d Medical College, N e w Delhi.
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