Charles E. Ahlfors

Toward Understanding Kernicterus: A Challenge to Improve the Management of Jaundiced Newborns

PURPOSE. We sought to evaluate the sensitivity and specificity of total serum bilirubin concentration (TSB) and free (unbound) bilirubin concentration (Bf) as predictors of risk for bilirubin toxicity and kernicterus and to examine consistency between these findings and proposed mechanisms of bilirubin transport and brain uptake. METHODS. A review of literature was undertaken to define basic principles of bilirubin transport and brain uptake leading to neurotoxicity. We then reviewed experimental and clinical evidence that relate TSB or Bf to risk for bilirubin toxicity and kernicterus. RESULTS. There are insufficient published data to precisely define sensitivity and specificity of either TSB or Bf in determining risk for acute bilirubin neurotoxicity or chronic sequelae (kernicterus). However, available laboratory and clinical evidence indicate that Bf is better than TSB in discriminating risk for bilirubin toxicity in patients with severe hyperbilirubinemia. These findings are consistent with basic pharmacokinetic principles involved in bilirubin transport and tissue uptake. CONCLUSIONS. Experimental and clinical data strongly suggest that measurement of Bf in newborns with hyperbilirubinemia will improve risk assessment for neurotoxicity, which emphasizes the need for additional clinical evaluation relating Bf and TSB to acute bilirubin toxicity and long-term outcome. We speculate that establishing risk thresholds for neurotoxicity by using newer methods for measuring Bf in minimally diluted serum samples will improve the sensitivity and specificity of serum indicators for treating hyperbilirubinemia, thus reducing unnecessary aggressive intervention and associated cost and morbidity.

Renal calcification in preterm infants: Pathophysiology and long-term sequelae

We examined the clinical course of 17 preterm infants with chronic lung disease who received loop diuretics and developed nephrocalcinosis; nine of them were followed for up to 4.5 years. The mean gestational age was 26.8 weeks (SD 2.2 weeks), and mean birth weight was 830 gm (SD 276 gm). The diagnosis of renal calcification was made at a mean age of 12 weeks (SD 6.5 weeks) by abdominal x-ray examination, screening abdominal ultrasound studies or, both. Calcification was associated with both furosemide therapy and the presence of multiple potential risk factors. Renal calcification, length, and function were subsequently evaluated in nine patients at a mean age of 21.3 months (SD 15.3 months). Improvement in calcification occurred in five patients, with total resolution in four. Renal length, determined by ultrasound examination and corrected for body length, was normal in 17 of 18 kidneys. Serum creatinine values and calculated glomerular filtration rates were abnormal in four of nine patients. We conclude that renal calcification in preterm infants is associated with multiple risk factors, including furosemide usage, and tends to diminish during the first year of life. However, renal function may remain compromised in some patients.

Unbound (Free) Bilirubin: Improving the Paradigm for Evaluating Neonatal Jaundice

BACKGROUND The serum or plasma total bilirubin concentration (B(T)) has long been the standard clinical laboratory test for evaluating neonatal jaundice, despite studies showing that B(T) correlates poorly with acute bilirubin encephalopathy (ABE) and its sequelae including death, classical kernicterus, or bilirubin-induced neurological dysfunction (BIND). The poor correlation between B(T) and ABE is commonly attributed to the confounding effects of comorbidities such as hemolytic diseases, prematurity, asphyxia, or infection. Mounting evidence suggests, however, that B(T) inherently performs poorly because it is the plasma non-protein-bound (unbound or free) bilirubin concentration (B(f)), rather than B(T), that is more closely associated with central nervous system bilirubin concentrations and therefore ABE and its sequelae. CONTENT This article reviews (a) the complex relationship between serum or plasma bilirubin measurements and ABE, (b) the history underlying the limited use of B(f) in the clinical setting, (c) the peroxidase method for measuring B(f) and technical and other issues involved in adapting the measurement to routine clinical use, (d) clinical experience using B(f) in the management of newborn jaundice, and (e) the value of B(f) measurements in research investigating bilirubin pathochemistry. SUMMARY Increasing evidence from clinical studies, clinical experience, and basic research investigating bilirubin neurotoxicity supports efforts to incorporate B(f) expeditiously into the routine evaluation of newborn jaundice.

Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population

Objective:The objective of this study was to determine if plasma unbound or ‘free’ bilirubin concentration (Bf) measured during the first 30 days of life is associated with subsequent abnormal hearing screening testing by automated auditory brainstem response (AABR) in a diverse population of newborns.Study Design:An observational study of newborns receiving AABR, plasma total bilirubin concentration (TBC) and Bf measurements and without underlying conditions known to affect hearing was conducted. Logistic regression was used to determine associations between abnormal AABR and Bf or TBC. The impacts of a variety of clinical factors on the regression model were also assessed.Result:A total of 191 patients with birth weights and gestations ranging from 406 to 4727 g and 24 to 42 weeks, respectively, were studied. Among them, 175 (92%) had normal (bilateral PASS) AABR and 16 had abnormal AABR (6 had unilateral REFER AABR, and 10 had bilateral REFER AABR). Mean TBC was not significantly different in babies with normal or abnormal AABR, but mean Bf was greater in the latter group (1.76 versus 0.93 μg per 100 ml, respectively, P=0.012). Bf, but not TBC, was associated with an abnormal AABR (Bf adjusted odds ratio 3.3, 95% CI 1.8 to 6.1). Comparing receiver-operating characteristics curves, the Bf/TBC ratio was a better predictor of an abnormal AABR than Bf alone. Intraventricular hemorrhage was the only confounding clinical variable.Conclusion:An abnormal AABR is associated with an elevated Bf or Bf/TBC ratio, but not the TBC alone. The prevalence of bilirubin neurotoxicity as a cause of audiological dysfunction may be underestimated if the TBC alone is used to assess the severity of newborn jaundice.

Influence of clinical status on the association between plasma total and unbound bilirubin and death or adverse neurodevelopmental outcomes in extremely low birth weight infants

Objectives:  To assess the influence of clinical status on the association between total plasma bilirubin and unbound bilirubin on death or adverse neurodevelopmental outcomes at 18–22 months corrected age in extremely low birth weight infants.

Factors Affecting the Binding of Bilirubin to Serum Albumins: Validation and Application of the Peroxidase Method

The unbound “free” bilirubin concentration (Bf), not the total bilirubin concentration, is the critical determinant of cellular uptake and toxicity of bilirubin. We compared Bf measured by a modified peroxidase method with published data obtained with ultrafiltration and examined conditions that affect the affinity (KF) of human (HSA) and bovine (BSA) serum albumin for bilirubin. The peroxidase and ultrafiltration methods yielded similar KF values that decreased with increasing HSA concentration and the presence of 50 mM chloride. When related to ionic strength, inhibition of BSA-bilirubin binding by chloride, bromide, and sulfate were similar, whereas phosphate buffer had a smaller effect. KF was lower at 37°C than at 25°C for HSA but not for BSA. KF for BSA was similar at pH 7.4 and 8.0. BSA and FCS had similar binding properties. The close agreement of Bf and KF values determined by the peroxidase method with published results obtained by ultrafiltration validates both methods and supports the use of the peroxidase method as a practical technique for measuring Bf under steady state conditions in minimally diluted serum or culture medium.

Bilirubin photooxidation products in the urine of jaundiced neonates receiving phototherapy.

Summary: Bilirubin-IXα photooxidation products were detected by high performance liquid chromatography in the urine of neonates undergoing phototherapy for hyperbilirubinemia. The in vivo photoproducts were identified by chromatographic comparison with authentic synthetic standards using two complementary methods. Bilirubin photooxidation products were not detected in urine from jaundiced infants not receiving phototherapy. The specific photoproducts identified in the urine include propentdyopents, hematinic acid imide and its hydrolysis product (3-carboxy-2-methyl-2-hexenedioic acid), and the hydrolysis product (2-vinyl-3-methyl-maleic acid) of methylvinylmaleimide. Their total urinary concentrations were low (0.2–0.9 mg/dl) during phototherapy. These observations show that photooxidation of bilirubin clearly does occur during phototherapy. They are consistent with the view that, although photooxidation is not the major photochemical event associated with phototherapy, it can and clearly does occur concurrently with photoisomerization.

Unbound Bilirubin Concentration is Associated With Abnormal Automated Auditory Brainstem Response for Jaundiced Newborns

OBJECTIVE. This study was conducted to determine whether incidental jaundice affects automated auditory brainstem response results. METHODS. We reviewed the medical charts of jaundiced newborns of ≥34 weeks of gestation who underwent automated auditory brainstem response testing within 4 hours of plasma total bilirubin concentration and unbound bilirubin concentration measurements. We tested the hypothesis that the likelihood of abnormal automated auditory brainstem response results would increase as total bilirubin and unbound bilirubin concentrations increased. RESULTS. Forty-four infants with proximate total bilirubin concentration, unbound bilirubin concentration, and automated auditory brainstem response measurements were identified, and 4 (9%) had bilateral refer automated auditory brainstem response results. The mean total bilirubin concentration of 21.4 mg/dL (SD: 4.0 mg/dL; range: 14.4–29.5 mg/dL) for the 40 infants with bilateral pass automated auditory brainstem response results was not significantly different from that of 23.0 mg/dL (range: 14.9–33.1 mg/dL) for the 4 infants with bilateral refer automated auditory brainstem response results. However, the mean unbound bilirubin concentration of 1.32 μg/dL (range: 0.22–2.99 μg/dL) for the 40 infants with bilateral pass results was significantly lower than the mean of 2.62 μg/dL (range: 0.88–4.41 μg/dL) for the 4 infants with bilateral refer results. Logistic regression showed that increasing unbound bilirubin concentrations but not increasing total bilirubin concentrations were associated with of bilateral refer automated auditory brainstem response results. CONCLUSIONS. The probability of bilateral refer automated auditory brainstem response results increases significantly with increasing unbound bilirubin concentrations but not with increasing total bilirubin concentrations. Because unbound bilirubin concentrations are also more closely correlated with bilirubin neurotoxicity than are total bilirubin concentrations, bilateral refer automated auditory brainstem response results for jaundiced newborns may indicate increased risk of bilirubin neurotoxicity, in addition to the possibility of congenital deafness.

Displacement of bilirubin from human albumin bythree diuretics

The interaction of three diuretics with bilirubin-albumin complexes was studied using the peroxidase assay, erythrocyte uptake, and sephadex gel filtration. On a molar basis, each diuretic was as potent or more potent than sulfisoxazole in displacing bilirubin from albumin. Furosemide and ethacrynic acid, when used at the recommended dosage (1 mg/kg), would probably not produce a significant increase in free bilirubin in most infants. Chlorothiazide could introduce a significant risk to jaundiced infants because of the higher dosage required.

The pathochemistry of kernicterus

The stoichiometry of bilirubin--albumin interaction has been analyzed and quantitated in several recent studies, confirming that albumin binding of bilirubin obeys the law of mass action [4, 5, 14, 16, 26, 36, 43, 46, 61, 65, 73, 92, 111]. These studies provide a basis for interpreting bilirubin transport, cell uptake and toxicity from physicochemical and pharmacologic perspectives [35, 42, 58, 59]. In this report, we propose a model of the pathogenesis of kernicterus which views serum albumin and tissue as competing with each other for binding the miscible bilirubin pool. Evidence is presented to show that bilirubin normally binds reversibly to cellular membranes and certain soluble enzymes just as it does to albumin; the unbound bilirubin concentration is the driving force for both albumin and tissue binding. We propose that albumin binding is determined by the concentration of free bilirubin anion (which is essentially unaffected by physiologic pH changes), and that tissue binding is mainly determined by the concentration of free bilirubin acid (which is greatly influenced by pH). When bilirubin--tissue complexes are formed, essential cell functions may be inhibited, producing cellular acidosis, irreversible intracellular aggregation of bilirubin, and cell death. In developing this argument, we will sequentially discuss relevant features of bilirubin chemistry, the binding of bilirubin to albumin, the formation of bilirubin--tissue complexes, bilirubin toxicity, alternative viewpoints of bilirubin transport, and, finally, the implications of this model to the clinical management of jaundiced infants. It should be emphasized that this paper is an attempt to analyze bilirubin transport and toxicity using basic chemical principles; it is an extension of previously published proposals [17, 77], and will undoubtedly require further modification as additional experimental data becomes available.