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Charles E Ahlfors

Publications and source records attributed to Charles E Ahlfors.

9 recordsLinked to original sources
PubMed2006

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 degrees C than at 25 degrees 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.

Animals↗
PubMed2006

Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns.

OBJECTIVES: To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl(-)) on plasma unbound bilirubin (B(f)) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns. DESIGN AND METHODS: B(f) was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with B(f) measured at a 2-fold sample dilution using a FloPro Analyzer. B(f) was measured at two peroxidase concentrations to determine whether the peroxidase steady state B(f) (B(fss)) measurements were significantly less than the equilibrium B(f) (B(feq)), in which case it was necessary to calculate B(feq) from the two B(fss) measurements. B(f) was also measured before and after adding 100 mmol/L Cl(-) to the UB Analyzer assay buffer. RESULTS: B(feq) at the 42-fold dilution was nearly 10-fold less than but it correlated significantly with B(feq) at the 2-fold dilution (mean 8.2+/-5.2 nmol/L versus 73.5+/-70 nmol/L, respectively, p<0.0001; correlation r=0.6). The two UB Analyzer B(fss) measurements were significantly less than B(feq) in 42 of 74 (57%) samples, and Cl(-) increased B(feq) in 66 of 74 (89%) samples by a mean of 82+/-67%. CONCLUSIONS: B(fss) measured by the UB Analyzer at the standard 42-fold sample dilution using assay buffer without Cl(-) and a single peroxidase concentration is significantly less than the B(feq) in undiluted plasma. Accurate B(f) measurements can be made only in minimally diluted serum or plasma.

Analytic Sample Preparation Methods↗
PubMed2006

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.

Bilirubin↗
PubMed2005

Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics.

BACKGROUND: Measuring plasma unbound bilirubin concentration by the peroxidase test is useful in the management of jaundiced newborns. However, the commercially available peroxidase technology is manual, and the unbound bilirubin may be seriously underestimated at the 42-fold sample dilution and single peroxidase concentration used. We investigated improving the test by adapting it to Zone Fluidics, which is a system for automating reactant handling that requires small sample volumes and dilution. METHODS: A computer-directed Zone Fluidics system was constructed using small diameter tubing to connect in series a water-surfactant reservoir, a bi-directional pump, a multiport selection valve to which peroxidase test reactants (45 mul of sample) are attached with one port open to air, and a spectrophotometer flow cell. Test reactants and air are sequentially aspirated through the valve into the tubing connecting the pump and valve to form a reactant "zone" surrounded by air. The zone is advanced to the spectrophotometer flow cell where total and unbound bilirubin are determined (37 degrees C) from the absorbance at 460 nm at a 2-fold sample dilution and 4 peroxidase concentrations. Imprecision was assessed in artificial controls and newborn plasma. Plasma results were compared with those obtained using the commercial method. RESULTS: The CV for unbound bilirubin in the various controls ranged from 11% to 38% (within day) and 12% to 27% (between days). Triplicate CV measurements for newborn plasma measurements ranged from 0.6% to 31% (mean 11%, n=47). Mean unbound bilirubin by Zone Fluidics was 5-fold higher than that by the commercial method. CONCLUSION: Zone Fluidics can be used to automate the peroxidase test and overcome many of the limitations of the commercially available peroxidase technology.

Bilirubin↗
PubMed2005

Evaluation of a model for brain bilirubin uptake in jaundiced newborns.

A model for brain bilirubin uptake (BBU) predicts that BBU in jaundiced newborns typically depends on the plasma total bilirubin concentration (TBC) and the bilirubin-albumin dissociation rate constant (k1) rather than the unbound bilirubin (Bf). The model's validity was tested by 1) evaluating its requirement that k3>>>k2, where k3 and k2 are the rate constants for BBU and Bf-albumin association, respectively, and 2) determining whether the calculated BBU is <or=5% of the bilirubin production rate, the approximate BBU expected if brain bilirubin levels are <1% of the miscible bilirubin pool as reported in the literature. The model was investigated using peroxidase test measurements of TBC, Bf, k1, and k2 from 185 jaundiced newborns. Mean k2 was compared with the reported k3 value of 0.08/s. BBU calculated from TBC and k1 was expected to be <or=0.005 microg/kg/s given the reported bilirubin production rate of 0.1 microg/kg/s. BBU calculated using Bf was also compared with the bilirubin production rate. The mean k2 of 8.9 L/micromol/s was greater than k3, and the mean BBU of 0.72 microg/kg/s exceeded the expected range of <or=0.005 microg/kg/s. However, mean BBU using Bf (0.00073 microg/kg/s) was within the expected range. A mathematical model calculating BBU as a function of TBC and k1 could not be validated. BBU calculated from Bf is consistent with the observation that <1% of the miscible bilirubin pool is distributed in the brain.

Bilirubin↗
PubMed2004

Effect of ibuprofen on bilirubin-albumin binding.

Ibuprofen is used for closing the ductus arteriosus in premature newborn infants. Ibuprofen interferes with bilirubin-albumin binding and increases the unbound bilirubin in pooled newborn plasma to levels similar to those produced by sulfisoxazole, a drug that causes kernicterus in premature newborn infants.

Albumins↗
PubMed2004

Bilirubin-albumin binding and neonatal jaundice.

Bilirubin-albumin binding weakens the correlation between the plasma total bilirubin concentration (TBC) and bilirubin encephalopathy (kernicterus), which has led to considerable interest over the years in measuring binding as part of the evaluation of jaundiced newborns. Despite development of several bilirubin-albumin binding tests, technical, ethical, and logistical factors have prevented the prospective studies needed to validate their routine clinical use. Consequently, it has been necessary to adopt aggressive exchange transfusion criteria based on the TBC that require the unnecessary treatment of large numbers of babies to prevent the rare case of kernicterus. Recently, early hospital discharge and arbitrary relaxation of exchange transfusion criteria have resulted in a resurgence of kernicterus. This resurgence and studies showing that nonalbumin bound or "free" bilirubin (Bf) correlates better than the TBC with bilirubin toxicity have rekindled interest in bilirubin binding tests. Technological advances in the measurement of Bf provide a convenient and economical means for integrating Bf measurements into routine clinical practice by determining the bilirubin-albumin binding parameters of various newborn populations.

Bilirubin↗