May-August, 2011/Vol 31/Issue 2

Personal Opinion

Biochemical Markers in Perinatal Asphyxia

Naithani M1, Simalti AK2 Dr. Manisha Naithani, MBBS, MD, Associate Professor, Department of Biochemistry, Sri Guru Ram Rai Institute of Medical Sciences, Deheradun, India, 2Dr. Ashish Kumar Simalti, MBBS, MD. Graded Specialist in Paediatrics, Military Hospital, Agra.
1

Address for correspondence: Dr. AK Simalti, E-mail: ashishsimalti@rediffmail.com

Abstract
Early assessment of the severity of an acute cerebral lesion secondary to hypoxia-ischemia or other pathologic conditions may provide a very useful basis for preventive or therapeutic decisions in pediatric patients. In the present review, we discuss the diagnostic and prognostic value of a series of biochemical parameters, with special reference to the diagnosis of neonatal HIE. Currently many specific biochemical markers of brain injury are being investigated to assess regional brain damage after perinatal asphyxia in neonates of which serum protein S-100, brain-specific creatine kinase, neuron-specific enolase, IL6 and urinary uric acid levels appear promising in identifying patients with a risk of developing hypoxic-ischemic encephalopathy. Whether detection of elevated serum concentrations of these proteins reflects long-term neurodevelopmental impairment remains to be investigated. Key words: S-100, Brain specific creatine kinase, neuron specific enolase, IL6, urinary uric acid, hypoxic ischaemic cerebral injury.

Introduction
ypoxic-ischemic cerebral injury that occurs during the perinatal period is one of the most commonly recognized causes of severe, long-term neurological decits in children. Hypoxic-ischemic encephalopathy (HIE) of the newborn occurs with the incidence of 14/10001. Between 20 % and 50 % of newborn infants affected by perinatal brain injury die during the newborn period, and 2560 % of the survivors suffer from permanent neurodevelopmental handicaps, including cerebral palsy, seizures, mental retardation, and learning disabilities2,3. Early assessment of the severity of an acute cerebral lesion secondary to hypoxiaischemia or other pathologic conditions may provide a very useful basis for preventive or therapeutic decisions in pediatric patients. Some authors have suggested that biochemical indicators may be more effective than the results of clinical evaluation, Apgar score, pH in cord blood, electroencephalographic and neuroimaging data4. Among the earliest biochemical markers of brain hypoxia in the CSF and/or plasma studied were lactate which accumulates in hypoxic cells due to glycolysis5. Among enzymes released from the cells during brain

hypoxia, the rst studied were LDH with isoenzymes, creatine-kinase (particularly its brain-specic isoenzyme CK-BB) and adenylate kinase. In the present review, we discuss the diagnostic and prognostic value of a series of biochemical parameters, with special reference to the diagnosis of neonatal HIE.

S100B
The term S100B refers to members of a multigenic family of calcium-modulated proteins (S100 proteins), mostly of low molecular mass (10,000 Da), At present, at least 20 proteins have been identied as belonging to the S100 protein family, the members of which are characterized by the presence of a pair of so-called EF-hand (i.e., helix-loop-helix) calcium-binding motifs6. Interestingly, the S100 proteins are highly conserved in amino acid composition among vertebrate species, and S100-like proteins have also been immunologically detected in planarians and spinach leaves S100B, a homodimer of a subunit ( subunit) that constitutes the bulk of the fraction originally isolated from brain extracts, was regarded for more than a decade as specic to the nervous system7. In the nervous system,

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the protein appears to be most abundant in glial cells, although its presence in neuronal subpopulations has also been reported9. The biological role of this protein within the cell populations that contain it has not been completely elucidated, although many hypotheses have been formulated, including the inhibition of protein phosphorylation, inhibition of cytoskeletal constituent assembly, stimulation of enzyme activities, and interaction with transcription factors9. An extracellular biological role has also been hypothesized for S100B, which secreted by astrocytes as a cytokine could have a neurotrophic effect during both development and nerve regeneration at physiologic (nanomolar) concentrations, but at high (micromolar) concentrations could be neurotoxic, participating in the pathophysiology of neurodegenerative disorders10,11. In this respect, it could be relevant that S100B is coded on the long arm of chromosome 21 (21q22.3), which is also involved in the translocation that causes Down syndrome12. An increase in gene expression for the protein has also been related to the neurodegenerative processes associated with both Down syndrome and Alzheimer disease, on the basis of ndings indicating that -amyloid stimulates the synthesis of S100b, whereas -amyloid precursor protein increases in cultures exposed to S100B13. Longitudinal S100B protein measurements in urine soon after birth are a useful tool to identify which asphyxiated infants are at risk of long-term neurologic sequelae. In a study by Diego Gazzolo, et al S100B protein levels were signicantly higher in samples collected at all monitoring times from new borns with abnormal neurologic ndings on follow-up. An S100B concentration cutoff of 0.28 g/L at rst urination had a sensitivity of 100% and a specicity of 87.3% for predicting the development of abnormal neurologic ndings on follow-up. The sensitivity and specicity of measurements obtained between 12 and 72 hours were up to 100% and 98.2%, respectively14.

serum levels were studied serially during the perinatal period in preterm neonates with perinatal asphyxia as markers of glial and neuronal damage respectively. The results of this study showed that neonates with severe asphyxia at any time had signicantly more elevated mean serum levels of both markers compared to the group with mild asphyxia and to the control group (p<0.05). The values of control group were also signicantly lower in comparison with that of mild asphyxia. In neonates with severe asphyxia, NSE values decreased constantly from birth to the seventh day of life, while PS-100 showed a different pattern increasing progressively between 3 h and 7 days. In neonates with mild asphyxia serum values of both markers showed decreasing levels through the study period17. Tekgul and colleagues also reported that the levels of NSE in cerebrospinal uid (CSF) were signicantly higher in cases of moderate to severe HIE vis--vis mildly affected infants. However, they found that though the CSF levels of NSE correlated relatively strongly with the degree of HIE, the serum NSE levels did not show signicant correlation with the same18. Also, Verd Prez A, et al found that, the presence of elevated NSE values in blood after perinatal asphyxia can be a sensitive indicator of conspicuous brain damage. The combined information provided by the severity of the encephalopathy together with the blood NSE values, has high predictive value for neurological outcome. NSE levels in the group of neonates who died or developed neurological sequelae were signicantly higher than NSE values in the group of neonates who were neurologically normal at follow-up. There were not differences between NSE values in the group of neonates who developed neurologically normal and the control group. Sensitivity and specicity of blood NSE as predictor of poor outcome were, respectively, 100% and 78%. The combined specicity for blood NSE together with a moderate/severe encephalopathy was 95%19. In a similar study by E Thornberg NSE was signicantly increased in the CSF of infants with HIE (median value 25.4 pug/l) compared with control infants (10.0 pug/I). Infants with the highest concentrations died20. So we can say that blood levels of NSE may or may not be reliable indicator in view of contradicting reports of studies but CSF levels are reliable predictors.

Neuron Specic Enolase

Neuron-Specic Enolase (NSE), a homodimer of the gamma form of enolase, is localized in the cytoplasm of neurons and cells of neuronal origin. NSE is an enzyme which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway, and also the reverse reaction in gluconeogenesis15. Perhaps not surprisingly since neurons require a great deal of energy they are very rich in glycolytic enzymes such a GAPDH and NSE. Its presence in cerebrospinal uid and blood is attributed to cell destruction. Serum NSE level is a marker for neuronal damage after traumatic brain injury, tumor, and neurodegenerative diseases16. In a study by Giuseppe, Distefano et al Protein S-100 (PS-100) and Neuron Specic Enolase (NSE)

IL6
Experimental studies suggest that cytokinemediated inammatory reactions are important in the cascade leading to hypoxic ischemic brain injury. In their study Tekgul et al found that the levels of IL-6 in

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both serum and CSF were signicantly higher in infants with moderate to severe HIE than in the mildly affected infants18. IL-6 in CSF best predicted the outcome at age 2 years. A cutoff value of 25.9 pg/mL was 100% sensitive and 79% specic for an adverse outcome, and had positive and negative predictive values of 100% and 86%, respectively. Svman, Karin in a study measured the content of pro- and anti -inammatory cytokines in CSF of asphyxiated and control infants. The concentrations of IL-1, IL-8, IL-10, tumor necrosis factor (TNF)-, and granulocyte/monocyte colony-stimulating factor (GMCSF) were determined. The concentration of IL-6 was higher in asphyxiated than in control. There was also a signicant relationship between IL-6 and the degree of HIE, and the nal outcome21. Xanthou also reported a rise in serum levels of IL-6, IL-1 and sICAM-1 in perinatally asphyxiated and infected neonates22. Martn Ancel and colleagues concluded in their study that the magnitude of IL-6 response in the CSF after perinatal asphyxia is related to the severity of neonatal HIE, to brain damage, and to the neurological outcome23. Various other centres have also reported comparable results to suggest that IL-6 might play a role in neonatal hypoxic-ischemic brain damage24,25.

to asses the validity of urinary uric acid as a marker of the degree of perinatal asphyxia. The ratio of urinary uric acid to creatinine (UA/Cr) in urine specimens obtained after birth were signicantly higher in asphyxiated infants who were identied by an Apgar scores 5 at 5 min and/ or an umbilical cord blood pH 7.2, and a base decit 12 meq/1. Also, within the perinatal asphyxia group, a signicant correlation was found between the UA/Cr ratio and the asphyxia score31. During reoxygenation after hypoxia, hypoxanthine is oxidized by the action of xanthine oxidase, forming xanthine. This reaction generates free radicals (superoxide anion, hydrogen peroxide, hydroxyl radical), which may in turn cause tissue damage. The xanthine-oxidase inhibitor allopurinol (ALLO) reduces free radical formation, thereby limiting the amount of hypoxia-reperfusion damage. Animal and human studies suggest that administration of ALLO immediately prior to delivery in case of suspected foetal asphyxia might reduce hypoxic-ischaemic encephalopathy. Kaandorp et al have proposed a study on effect of antenatal injection of allopurinol32. Chen HJ, et al studied the validity of urinary uric acid as a marker of perinatal asphyxia in term and premature infants. The urinary UA/Cr was signicantly higher in term and preterm infants with perinatal asphyxia than in their unasphyxiated counterparts. The urinary UA/Cr in premature infants was signicantly higher than in term infants. When the urinary UA/Cr was > 0.95, perinatal asphyxia was identied with a sensitivity of 80% and a specicity of 71% in term infants. They concluded that the urinary UA/Cr may be used as an additional marker of perinatal asphyxia in term and premature infants. In comparison with other markers it is a simple, quick, and inexpensive way to detect hypoxic episodes in a neonatal intensive care unit within 24 hours after birth33.

Urinary Uric Acid

Tissue hypoxia is a recognized cause of adenine nucleotide depletion, which is accompanied by the appearance of intermediate catabolites. These intermediates are used as metabolic markers of ATP depletion in tissues. Oxypurines (hypoxanthine and xanthine) are the nal products of ATP degradation followed by uric acid formation26,27. Under normal conditions, about 90% of the hypoxanthine is reconverted to purine nucleotides by the action of HGPRT, restoring the ATP pool. Hypoxanthine is also converted to xanthine without the production of free radicals by the action of xanthine dehydrogenase, using nicotinamide adenine dinucleotide as substrate. During hypoxia, cellular ATP concentrations drop and there is accelerated degradation of adenosine monophosphate (AMP) to hypoxanthine28. Several experimental studies have indicated some xanthine oxidase activity in cerebral capillaries, and raised uric acid levels after periods of hypoxia29. Perlman and Risser determined serum uric acid levels over the rst 2 days post-partum in premature neonates at risk for intraventricular hemorrhage or periventricular leukomalacia, and found that levels were signicantly raised in neonates who subsequently developed either of these complications30. David Bader, David Gozal et al conducted a study

Creatinine Kinase
Creatinine kinase BB (CK), a key enzyme in the energy metabolism of cells with intermittently high and uctuating energy requirement (e.g. skeletal and cardiac muscle, neural tissues like brain and retina) catalyzes the reversible transfer of the phosphoryl group from phosphocreatine to adenosine diphosphate, to regenerate adenosine triphosphate (ATP). The distinct isoenzyme-specic localization of CK isoenzymes in brain suggests an important function for CK in brain energy and points to adaptation of the CK system to special energy requirements of different neuronal and glial cell types. The determination of CK and its histospecic isoenzymes in serum is a standard technique for evaluating disorders that affect the

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myocardium, the skeletal muscle and the central nervous system (CNS). Hence, serum and CSF brain derived CK (CK-BB) isoenzyme determination in neonates, has been suggested to be of value for predicting the degree of neuronal damage and even developmental prognosis. V J Ruth studied serum CK-BB activity on the rst day of life in acutely asphyxiated infants, infants born after high risk pregnancies (pre-eclampsia/ intrauterine growth retardation, or both), and very low birth weight infants. Neuro-developmental evaluation was carried out at 2.2-2.5 years. They showed that infants who died with brain injury had signicantly higher CK-BB activity than infants with normal outcomes. CK-BB in infants with cerebral palsy and mild motor impairment were similar to controls34. Cuestas observed that in healthy neonates serum CK-BB activity declines rapidly after birth, reaching a stable value after 6h-15 days, whereas neonates with severe asphyxia and neurologic damage showed a rapid increase in CK-BB concentration35. Walsh and colleagues monitored CK-BB activity in cord blood over 6-80h post-partum, in normal and severely asphyxiated neonates, nding that CK-BB activities 2.5 standard deviations higher than the control-group correctly predicted subsequent neurologic abnormality in 17 (77%) of the 22 asphyxiated neonates, and in 11 (92%) of the 12 asphyxiated neonates who survived the neonatal period. These authors concluded that serum CK-BB activity determined in cord blood between 6 and 12 h post-partum is an effective predictor of neurological prognosis following severe neonatal asphyxia36. Other studies have looked at CK-BB in CSF. In their independent trials, Dalens37 and Talvik38 observed that CSF CK-BB levels determined on day three of life was an effective predictor of neurologic anomalies detected at 12 months of age. Nagdyman Nicole et al in a study examined CK-BB, protein S-100, and neuron-specic enolase in cord blood and 2, 6, 12, and 24 h after birth in 29 asphyxiated and 20 control infants. A combination of serum protein S-100 and CK-BB 2 hours after birth had the highest predictive value and specicity of predicting moderate and severe HIE. Cord blood pH and cord blood base decit increased the predictive values of protein S-100 and CK-BB39.

Acute CNS insults may lead to very high concentrations in CSF, probably because of the disintegration of astroglial cells, while moderate increases are observed in other clinical conditions such as Alzheimers disease, dementia secondary to multiple infarct, and infantile autism. GFAP has extensively been used as a marker of gliosis in neuropathology. HIE after perinatal asphyxia is a condition in which levels of GFAP might be elevated if brain cell damage occurs. In animal studies, the core of the hippocampus has been shown to be surrounded by a well-developed network of GFAP-positive cells providing an apparent protective shield. That shield was less developed in cultures from asphyxia-exposed animals40. In a study of twenty-two asphyxiated infants, Blennow et al. observed that the extremely high concentrations of GFAP and others brain-specic proteins in the CSF were mainly found in the group of babies that died. They concluded that the cerebrospinal uid levels of GFAP might be an important adjunct in the neonatal assessment of infants subject to perinatal asphyxia, and together with other neuronal or glial proteins, it might also help in dening temporal relationships in asphyxia41,42.

Conclusion
The diagnosis of perinatal insults currently relies on adequate documentation of general medical and obstetric factors and on radiologic and laboratory assessments. To facilitate therapeutic strategies, early identication of the infant at highest risk for developing seizures secondary to hypoxia ischemia or asphyxia is critical, particularly if novel but potentially toxic therapies currently under experimental investigation become available for clinical use. Currently many specic biochemical markers of brain injury are being investigated to assess regional brain damage after perinatal asphyxia in neonatesof which serum protein S-100, brain-specic creatine kinase, neuron-specic enolase, IL6 and urinary uric acid levels appear promising in identifying patients with a risk of developing hypoxic-ischemic encephalopathy. Whether detection of elevated serum concentrations of these proteins reects long-term neurodevelopmental impairment remains to be investigated.

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How to cite this article ? Naithani M, Simalti AK. Biochemical Markers in Perinatal Asphyxia. J Nep Paedtr Soc 2011;31(2):151-156.

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