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Special Feature NICHD Conference on Kernicterus: Research on Prevention of Bilirubin-Induced Brain Injury and Kernicterus: Bench-to-BedsideFDiagnostic Methods and Prevention and Treatment Strategies David K. Stevenson Ronald J. Wong Hendrik J. Vreman Antony F. McDonagh M. Jeffrey Maisels David A. Lightner

In July 2003, the National Institute of Child Health and Human Development (NICHD) organized a consensus conference, where a group of experts were invited to review and discuss the current state of knowledge regarding neonatal hyperbilirubinemia and identify areas in which where future research should be directed. This paper summarizes the presentations addressing the current methodologies for direct and noninvasive assessments of serum total bilirubin concentrations as well as prevention and treatment strategies for the management of neonatal hyperbilirubinemia. Journal of Perinatology (2004) 24, 521–525. doi:10.1038/sj.jp.7211124 Published online 6 May 2004

Neonatal jaundice is perhaps the most common problem encountered by infants in the transitional period after birth and therefore frequently seen by general pediatricians. However, its diagnosis and the diagnostic methods used are not universally uniform. Measurement of the concentration of serum total bilirubin (STB) is the mainstay both in the management of neonatal jaundice and the prevention of kernicterus. Therefore, high precision, reproducibility, and, above all, accuracy of the measurement are essential. Reports of large interlaboratory

Division of Neonatal and Developmental Medicine, Department of Pediatrics (D.K.S.,R.J.W.,H.J.V.), Stanford University School of Medicine, Stanford, CA, USA; Department of Medicine (A.F.M.), University of California, San Francisco, CA, USA; Department of Pediatrics (M.J.M.), William Beaumont Hospital, Royal Oak, MI, USA; and Department of Chemistry (D.A.L.), University of Nevada, Reno, NV, USA. Presented in part at the NICHD Conference, Research on Prevention of Bilirubin-Induced Brain Injury and Kernicterus-From Bench to Bedsideat Bethesda, MD; USA; June 2003. Address correspondence and reprint requests to David K. Stevenson, MD, Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stamford University, 750 Welch Road, Suite #315, Palo Alto, CA 94304-5731, USA.

variability in STB determinations are disturbing and call into question the intrinsic accuracy of current methodologies.1 An understanding of the structure, properties, and reactivity of bilirubin is essential to appreciating the problems and difficulties involved not only in accurately measuring the concentration of bilirubin and its metabolites in blood, but also in developing reliable analytical methods for identifying infants most at risk of developing kernicterus. First and foremost is the importance for clinicians to understand that bilirubin-IXa (4Z,15Z) is the unique molecular product of a two-step process beginning with heme. The first and rate-limiting step is catalyzed by heme oxygenase (HO) and requires molecular oxygen (O2) and NADPH, generated from the cytochrome P-450 system.2 This reaction yields biliverdin, carbon monoxide (CO), and iron (Fe2 þ ) in equimolar amounts. Biliverdin is reduced by biliverdin reductase, in the presence of NADPH, to form the single isomer bilirubin-IXa (4Z,15Z). It is the only bilirubin isomer entering the circulation after birth and is bound avidly by albumin. Bilirubin is then taken up into the liver, where it is conjugated by a specific isoform, uridine diphosphate glucuronosyltransferase (UGT1A1), to form mono- and diglucuronides. However, when these accumulate in blood, as in cholestatic jaundice, they undergo covalent binding to albumin, molecular rearrangement to give many isomers, and hydrolysis to unconjugated bilirubin.3–5 All of these species, except for unconjugated bilirubin, give the direct van den Bergh diazo reaction, which can be used to spectrophotometrically estimate, but not accurately quantitate, their concentrations in serum. Unconjugated bilirubin, on the other hand, gives rise to the indirect van den Bergh reaction, but a small fraction invariably reacts as direct bilirubinFan artifact that may produce misleadingly high STB levels. Serum from neonates with noncholestatic jaundice should in principle contain only bilirubinIXa (4Z,15Z isomer) along with a relatively very low concentration of direct-reacting bilirubin. In practice, the 4Z,15E isomer is invariably also present, formed by exposure of the patient (or the serum) to ambient or phototherapy light. The influence of this isomer on measurements of indirect or unbound bilirubin is not well established. In addition, standardization of STB measurements is a topic of great concern. Not only are commercial and NBS bilirubin standards often impure, but there is also tremendous

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interlaboratory variation in measurements of STB between laboratories.1 Although reproducibility within a particular method or a given laboratory may be adequate, accuracy, however, may not. Consequently, there is currently a great need for better standardization of bilirubin measurements as well as better education in the preparation of appropriate bilirubin standards and clinical specimens and in the interpretation of results. Another need is the development of ‘‘point of care’’ analytic methods and instruments for determining STB levels at the bedside or at home. These technologies are in various stages of development, but are not currently available for clinical use. Another important aspect of bilirubin physiology is the relationship between STB levels and bilirubin toxicity with respect to bilirubin–albumin binding. The measurement of reserve albumin-binding capacity can be used as an estimate of the likelihood that bilirubin, not bound to albumin (the so-called unbound or ‘‘free’’ bilirubin), will move from circulation into tissue. This movement of bilirubin is affected by the acid–base status of the neonate. With a decreasing pH, there is an increased likelihood of bilirubin deposition in the tissues and, in particular, the cells of the central nervous system. Another potential method for assessing the risk of bilirubin toxicity is the direct measurement of the concentration of ‘‘free’’ bilirubin. The peroxidase assay has been used for this measurement, but it has not been widely adopted or accepted in the United States. An improved and more useful peroxidase method has been developed, which might help facilitate studies probing the relationship of ‘‘free’’ bilirubin to neurologic sequelae.6 Currently, only limited data are available, but they support the association of ‘‘free’’ bilirubin levels with neurologic dysfunction or permanent injury. Large-scale studies are warranted before practical guidelines can be developed and implemented for the use and interpretation of ‘‘free’’ bilirubin measurements in the clinical setting. Neonatal jaundice is initially diagnosed by clinical inspection and recognition of a yellow color in the skin. However, the eye is relatively insensitive to yellow, making visual inspection unreliable. As a result, methods have been developed to measure skin color (and therefore estimate STB levels) by visual color-matching and by reflectance or transcutaneous colorimetry.7 The BiliChekt (Respironics, Norcross GA) and JM-103 Jaundice Meter (Minolta/ Hill-Rom Air-Shields, Hatboro PA)8 are transcutaneous bilirubinometers which have been shown to provide acceptable correlations between transcutaneous bilirubin (TcB) and STB concentrations in infants 35 weeks gestation. Measurements using the BiliChekt compare favorably with standard laboratory bilirubin measurements as well as with high-performance liquid chromatography determinations.9 The BiliChekt appears to work well in multiracial populations, but may be less accurate when STB levels are >15 mg/dl (257 mmol/l). The JM-103 is easier to use than the BiliChekt, but may be less accurate in infants with dark pigmentation. In these infants, the JM-103 generally overestimates 522

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STB levels, so that such errors would be unlikely to result in underdiagnosis of severe hyperbilirubinemia. Both the JM-103 and the BiliChekt appear to be less reliable when used on infants 15 mg/dl (257 mmol/l), or during and after phototherapy. The reliable use of TcB measurements in premature or low birth weight infants also needs to be fully explored. Nevertheless, TcB measurements can be used to identify infants who require additional investigation for a bilirubin level that is elevated for the infant’s age (in hours) and as a pre-discharge screening device to identify infants who are, or are not, at risk for the subsequent development of severe hyperbilirubinemia.10 The use of the BiliChekt or JM-103 should substantially reduce the number of invasive STB measurements obtained in these vulnerable neonates. Both devices seem to have acceptable diagnostic accuracy when used as screening devices for placing an infant in a risk category for follow-up10 or obtaining an STB concentration. The use of such technologies in the outpatient setting would be ideal. One of the more important causes of neonatal jaundice is increased bilirubin formation. Neonatal jaundice can be understood best by appreciating the dynamic balance between bilirubin production, which is normally increased in the newborn (two to three times higher in an infant compared with an adult), and bilirubin elimination, which is temporarily impaired after birth until the UGT-dependent conjugating system is fully mature and functional. A by-product of the first step is CO, which becomes first bound to hemoglobin in circulation as carboxyhemoglobin (COHb) and then is continually excreted in breath. Thus, measurements, corrected for inhaled CO, of COHb (COHbc) levels, end-tidal CO (ETCOc) concentrations, or the total body excretion rates of CO (VeCO), can be used as estimates of total bilirubin formation (TBF) in the neonate.11 These surrogates for TBF need to be understood in the context of total endogenous CO production. Approximately 86% of the CO produced endogenously comes from heme degradation (the senescence of red blood cells, ineffective erythropoiesis, and the degradation of other hemoproteins). Approximately 14% is believed to be derived from lipid peroxidation Journal of Perinatology 2004; 24:521–525

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and photo-oxidation, but these sources need much further study. In some pathologic conditions, the contribution from these latter two sources is variable, but could be quite high. Under normal conditions in the healthy term or near-term infant, the predominant source of CO is through heme degradation. Thus, estimates of CO production can be used as reasonable indices for TBF. The sensitivity, reproducibility, and accuracy of this approach have been mostly proven only under controlled laboratory conditions. The role of increased bilirubin production in transitional hyperbilirubinemia and its predominance as a cause of pathologic jaundice are well established in infants with hemolytic conditions, infants of diabetic mothers, and infants with polycythemia and closed-space bleeding. A population-based distribution of bilirubin production, as estimated by the ETCOc, has been described.11 High producers of bilirubin (i.e. high age-adjusted ETCOc levels) are likely to have some type of hemolytic process. On the other hand, infants with hyperbilirubinemia, but normal bilirubin production rates (i.e. normal age-adjusted ETCOc levels), probably have defective conjugation, as seen in Gilbert’s syndrome or in Asian infants carrying the G71R mutation in the UGT1A1 gene. In evaluating the jaundiced infant, determination of CO production is the only clinical measurement that provides direct information regarding the rate of bilirubin production. When combined with an hour-specific STB measurement, these two measurements can provide insight into the cause of jaundice and permit intervention directed at the particular pathophysiology (either increased production or impaired conjugation).11

SESSION IV: PREVENTION AND TREATMENT STRATEGIES Bilirubin-IXa, derived through the reduction of biliverdin-IXa, is a lipophilic molecule, insoluble in aqueous bicarbonate and alcohol, but soluble in chloroform, and is not excreted intact. It has a ridge-tile structure shaped like a half-open book and, under normal conditions, is conjugated in the liver by UGT to bilirubin monoglucuronides and thence to bilirubin diglucuronide. The two glucuronides are then secreted into bile. The structure of bilirubin makes it uniquely susceptible to interactions with particular wavelengths of light.12 When light interacts with a molecule creating a photo-excited state, subsequent de-excitation can occur by photoreactions (photochemistry), transfer of energy, or emission of photons. Photochemistry and vibrational deactivation predominantly occur when light interacts with bilirubin-IXa. Several photochemical reactions can occur, including the formation of E-photoisomers, cyclization to lumirubin, photodegradation, photodissociation products, and phototautomerization. However, E-photoisomers and lumirubin are the major products formed during phototherapy. Lumirubin is produced photochemically either directly from the 4Z,15Z isomer or Journal of Perinatology 2004; 24:521–525

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indirectly from the 4E,15Z isomer. Other products include a variety of photo-oxidation products. The principle of phototherapy for the treatment of hyperbilirubinemia is straightforward. As you can see bilirubin, it must absorb visible light. As you can see yellow bilirubin in jaundice, this means that blue light is absorbed. For phototherapy to be effective, it needs to be efficient with fast photoreactions, slow or no reversion to bilirubin, and fast elimination of photoproducts. Most important in the efficacy of phototherapy are light intensity, wavelength, and the body surface area exposed. Bilirubin absorbs in the visible between 350 and 520 nm with a peak at 450 to 475 nm. Since skin becomes more transparent towards longer wavelengths, the most effective light is in the blue or blue–green wavelengths. Ultraviolet light (A, B, or C) has no role in phototherapy. However, most current light sources emit small amounts of UVA radiation (315 to 400 nm, which is closest to the visible), which could have detrimental effects. Simple filtering (e.g. through plexiglass) prevents UV radiation from reaching the infant. The irradiance produced by white fluorescent tubes is significantly lesser than that of special blue tubes.13,14 The new phototherapy devices incorporating blue light-emitting diodes LEDs as a light source may be more effective than conventional fluorescent and halogen-based devices and do not emit UV light or infrared radiation. A number of side effects have been reported with use of traditional light sources.15 It would be interesting to determine if narrow wavelength band LED light sources do cause similar reactions. The relative toxicities of bilirubin photoproducts are presently unknown and require investigation. Photo-oxidation may be wavelength dependent and may have detrimental effects under some conditions, especially those encountered by very small, immature infants, whose skin are translucent. The generally beneficial application of light may, in fact, reduce the beneficial antioxidant effects of bilirubin at some level. Presently, little is known concerning the mechanism of phototherapy, and further investigations are needed to explore the long-term effects of the treatment, the influence of the environment on bilirubin photochemistry, and the optimal wavelengths and sources of applied light. A potentially important alternative therapy with good rationale is the use of metalloporphyrin (Mp) HO inhibitors for the treatment of hyperbilirubinemia. As bilirubin production is increased in most cases of pathologic jaundice (production rates are two to three times higher in an infant compared with an adult, even without an underlying pathologic condition), targeting the rate-limiting enzyme, HO, is a logical and attractive strategy. Mps are synthetic heme analogs with various substituents on the porphyrin macrocycle and different metals in the center of the ring. Besides inhibiting bilirubin production, they have also been shown to affect other processes and enzymes, including lipid peroxidation,16 photo-oxidation, nitric oxide synthase (NOS), and soluble guanylyl cyclase (sGC).17 After administration of Mps, 523

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uncatabolized heme appears in bile, to a degree which is proportional to the degree of inhibition of bilirubin production. However, there may be slight elevations in the heme content of tissues that might contribute to the induction of the HO-1 gene with consequent increased production of the enzyme, which, in turn, could mitigate the effectiveness of Mps if repeatedly administered. Another important side effect of some Mps is their potential for photosensitization, which has been demonstrated by in vitro and in vivo testing.18 The criteria for a potential antihyperbilirubinemic drug of this class would include potent HO inhibition, a biocompatible central metal, and negligible degradation, photoreactivity, and HO-1 upregulation. The latter characteristic has been evaluated using an HO-1-luc transgenic reporter mouse, which allows in vivo assessment of HO-1 transcriptional upregulation caused by the administration of Mps.19,20 This effect is important to evaluate because upregulation of this enzymatic system could have other associated biological consequences mediated through CO modulation of other enzymatic systems, including activation of sGC. At this time, several Mps offer promise as drug candidates for the treatment of transitional hyperbilirubinemia.21 Tin mesoporphyrin (SnMP) is a synthetic molecule with a non-biocompatible central metal and very high potency, and therefore can be used at doses

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that minimize its high photoreactivity. Although not orally absorbable, it has already been used in clinical studies with demonstrated efficacy and without apparent toxicity other than mild phototoxicity.22,23 Zinc bis glycol porphyrin (ZnBG) has a biocompatible central metal, has very high potency, and can also be used at a dose without phototoxicity. It is orally absorbable and may not inhibit sGC. Of all the compounds studied, ZnBG has the least effect on HO-1 transcription. Chromium mesoporphyrin (CrMP) is another Mp with a biocompatible central metal. It has high potency and no phototoxicity. It is orally absorbable and at low doses (15 mg/dl (257 mmol/l), when infants are under phototherapy, after phototherapy, and in premature infants The development of inexpensive, portable, and easy-to-use CO detection technology which is sensitive, reproducible, accurate, and unaffected by hydrogen for diagnosis of hemolysis The demonstration of the impact of CO detection on the management of neonatal jaundice, for example, altered surveillance, targeting for treatment, choosing of HO inhibitors The investigation of the relative importance of the various photochemical pathways and their effects in neonates using different light sources The study of the influences of the matrix and environment on bilirubin biochemistry, especially, with respect to toxicity The study of the optimal light sources for phototherapy with respect to wavelength, irradiance, and duration of body surface area exposure Elucidate and identify factors in infants with high STB levels who do not develop kernicterus Identify the bilirubin fraction which deposits in the skin and contributes to jaundice

HO, heme oxygenase; CO, carbon monoxide; TcB, transcutaneous bilirubinometry; STB, serum total bilirubin.

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Furthermore, new diagnostic approaches are improving the identification of infants at risk for pathologic jaundice. They also have begun to create a strong rationale for therapies which target patients for prevention and treatment strategies specific for their pathophysiology. Table 1 lists the areas of research that are needed as determined by those attending the consensus conference. Acknowledgements This work was supported by the National Institutes of Health, grants HL68703 (D.K.S.), HL58013 (D.K.S.), DK26307 (A.F.M.), and HD17779 (D.A.L.), and unrestricted gifts from the Mary L. Johnson, H. M. Lui, and Hess Research Funds (DKS).

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10. Bhutani VK, Johnson L, Sivieri EM. Predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and near-term newborns. Pediatrics 1999;103:6–14. 11. Stevenson DK, Fanaroff AA, Maisels MJ, et al. Prediction of hyperbilirubinemia in near-term and term infants. Pediatrics 2001;108:31–9. 12. McDonagh AF, Lightner DA. Phototherapy and the photobiology of bilirubin. Semin Liver Dis 1988;8:272–83. 13. Tan KL. Efficacy of fluorescent daylight, blue, and green lamps in the management of nonhemolytic hyperbilirubinemia. J Pediatr 1989;114: 132–7. 14. Vreman HJ, Wong RJ, Murdock JR, Stevenson DK. In vitro efficacy of an LED-based phototherapy device (neoBluet) compared to traditional light sources. Pediatr Res 2003;53:400A. 15. Aouthmany MM. Phototherapy increases hemoglobin degradation and bilirubin production in preterm infants. J Perinatol 1999;19:271–4. 16. Wong RJ, Vreman HJ, Stevenson DK. (Metallo)porphyrin inhibitors of heme oxygenase also inhibit lipid peroxidation (LP). Pediatr Res 2000;47:465. 17. Appleton SD, Chretien ML, McLaughlin BE, et al. Selective inhibition of heme oxygenase, without inhibition of nitric oxide synthase or soluble guanylyl cyclase, by metalloporphyrins at low concentrations. Drug Metab Dispos 1999;27:1214–9. 18. Vreman HJ, Ekstrand BC, Stevenson DK. Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatr Res 1993;33:195–200. 19. Zhang W, Contag PR, Hardy J, et al. Selection of potential therapeutics based on in vivo spatiotemporal transcription patterns of heme oxygenase-1. J Mol Med 2002;80:655–64. 20. Wong RJ, Abate A, Dennery PA, Vreman HJ, Contag CH, Stevenson DK. Intestinal absorption of metalloporphyrins and systemic effects on heme oxygenase. Pediatr Res 2003;53:400A. 21. Vreman HJ, Wong RJ, Stevenson DK. Alternative metalloporphyrins for the treatment of neonatal jaundice. J Perinatol 2001;21(Suppl 1):S108–13. 22. Valaes T, Petmezaki S, Henschke C, Drummond GS, Kappas A. Control of jaundice in preterm newborns by an inhibitor of bilirubin production: studies with tin-mesoporphyrin. Pediatrics 1994;93:1. 23. Martinez JC, Garcia HO, Otheguy LE, Drummond GS, Kappas A. Control of severe hyperbilirubinemia in full-term newborns with the inhibitor of bilirubin production Sn-mesoporphyrin. Pediatrics 1999;103:1–5.

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