Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics

Ahlfors, Charles E.; Marshall, Graham D.; Wolcott, Duane K.; Olson, Don C.; Overmeire, Bart Van

doi:10.1016/j.cca.2005.07.030

Cited by 38 publications

(41 citation statements)

References 34 publications

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“…Case series that include several neonates with peak TSB levels of greater than 40 mg per 100 ml and as high as 59.9 mg per 100 ml, 26,47 as well as peroxidase measurements of serum B F in human newborns lend credence to this speculation. Apparent serum B F levels in human newborns often greatly exceed 70 nM, 31,48 and a recent report notes an infant with kernicterus whose B F was found to be 130 nM 3 using a modified technique 48,49 that appreciably reduces but does not eliminate sample dilution effects (sample dilution may lead to an underestimation of B F 31, [48][49][50] ).…”

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confidence: 99%

Calculated free bilirubin levels and neurotoxicity

2009

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Although most bilirubin in the circulation is bound to albumin, a relatively small fraction remains unbound. The concentration of this 'free' bilirubin (B F ) is believed to dictate the biologic effects of bilirubin in jaundiced newborns, including its neurotoxicity. The threshold at which B F produces changes in cellular function culminating in permanent cell injury and cell death has been the subject of considerable debate. The objective of this study was to compare calculated central nervous system (CNS) B F levels in Gunn rat pups during (i) peak postnatal hyperbilirubinemia and (ii) sulfadimethoxine-induced acute bilirubin encephalopathy (ABE) previously reported from our laboratory with those predicted in human neonates with peak total serum bilirubin (TSB) levels of 35 mg per 100 ml (599 mmol l À1 ), a clinical cohort that often evidence moderate-to-severe adverse post-icteric neurodevelopmental sequelae. Homozygous j/j Gunn rat pups with neonatal hyperbilirubinemia due to a deficiency of the bilirubin conjugating enzyme uridine-diphosphate-glucuronosyl transferase 1A1 were studied along with non-jaundiced littermate heterozygous J/j controls. Sulfadimethoxine was used to displace bilirubin from albumin in hyperbilirubinemic j/j Gunn rat pups to increase their brain bilirubin content and induce ABE. Calculated Gunn rat CNS B F levels were determined as a function of genotype, sulfadimethoxine exposure and albumin-bilirubin binding constant. These data were compared with the human CNS B F predicted from the calculated serum B F in human neonates with a TSB of 35 mg per 100 ml as a function of albumin-bilirubin binding constant, albumin concentration and the assumption that at this hazardous bilirubin level there may be rapid equilibration of B F between serum and brain. There was a large gap between the upper limit of the calculated CNS B F 95% confidence interval (CI) range in non-jaundiced J/j pups (for example, 112 nM at k ¼ 9.2 l mmol À1 ) and the lower limit seen in the saline-treated hyperbilirubinemic j/j pups (556 nM at k ¼ 9.2 l mmol À1 ) as well as between the upper limit in saline-treated hyperbilirubinemic j/j pups (1110 nM at k ¼ 9.2 l mmol À1 ) and the lower limit seen in sulfadimethoxine-treated jaundiced j/j littermates (3461 nM at k ¼ 9.2 l mmol À1 ). There was considerable overlap and remarkable similarity between the predicted human CNS B F values at a TSB of 35 mg per 100 ml for a range of reported human serum bilirubin-albumin binding constants and albumin concentrations, and those calculated for saline-treated hyperbilirubinemic j/j Gunn rat pups. This exercise yielded strikingly similar apparent calculated neurotoxic B F levels for Gunn rat pups and human neonates rather than orders of magnitude differences that might have been predicted at the outset and add to a growing literature aimed at defining clinically germane neurotoxic B F thresholds.

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Calculated free bilirubin levels and neurotoxicity

2009

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“…TBC and B f were measured by the peroxidase method at a 42-fold sample dilution (NRN and CPMC samples) using a UB Analyzer (Arrows Co., Ltd., Osaka, Japan) [14] and at a 2-fold sample dilution (NRN samples only) using a FloPro Analyzer (Global FIA, Fox Island, WA) [22]. Limited sample volumes and instrument configuration issues made it impractical to use the UB Analyzer to measure B f at both sample dilutions.…”

Section: Methodsmentioning

confidence: 99%

“…Limited sample volumes and instrument configuration issues made it impractical to use the UB Analyzer to measure B f at both sample dilutions. The UB Analyzer and FloPro Analyzer differ primarily in reagent handling and light path lengths, and comparison TBC values are not statistically different between the instruments [22]. Since both instruments obtain B f from integrated rate equations that require measuring the change in TBC (change in absorbance at 460 nm corrected for haemoglobin) over time [14,22], it seems unlikely that any differences in B f with sample dilution would be solely due to intrinsic differences in the instruments.…”

Section: Methodsmentioning

confidence: 99%

“…The UB Analyzer and FloPro Analyzer differ primarily in reagent handling and light path lengths, and comparison TBC values are not statistically different between the instruments [22]. Since both instruments obtain B f from integrated rate equations that require measuring the change in TBC (change in absorbance at 460 nm corrected for haemoglobin) over time [14,22], it seems unlikely that any differences in B f with sample dilution would be solely due to intrinsic differences in the instruments. In addition, a decrease in B f with sample dilution was originally documented using a single instrument [16], and the phenomenon, which is not unique to B f [24], has been confirmed by ultrafiltration studies [18].…”

Section: Methodsmentioning

confidence: 99%

“…The UB Analyzer uses the peroxidase method [13,14] and measures B f at a 42-fold sample dilution, a single peroxidase concentration, and without chloride ion (Cl -) in the assay buffer [14]. Several studies using artificial serum and/ or pooled plasma samples indicate that B f may be affected by sample dilution [15][16][17][18], Clconcentration [18][19][20][21], and when measured by the peroxidase test, the peroxidase concentration used [17,22,23].…”

Section: Introductionmentioning

confidence: 99%

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

Ahlfors

Vreman

Wong

et al. 2007

Clinical Biochemistry

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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. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 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. Results-B NIH Public Access

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Quantitative determination of bilirubin by inhibition of chemiluminescence from lucigenin

et al. 2007

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Bilirubin is a metabolic breakdown product of blood haem, of great biological and diagnostic importance. A new chemiluminescence (CL) method has been developed for the quantification of bilirubin. The method is combined with the flow injection analysis (FIA) technique and based on the inhibition effect of bilirubin on the CL from the lucigenin-hydrogen peroxide system in an alkaline medium. Under the optimum conditions, the decreased CL intensity was proportional to the concentration of bilirubin, in the range 0.0585-58.47 microg/mL. The detection limit estimated from the calibration graph was about 7.8826 ng/mL. The relative standard deviation (RSD) of 10 parallel measurements (1 x 10(-4) mol/L bilirubin) was 2.5%. Recoveries of bilirubin were found to fall in the range 94-97.5% using control sera. The method is interference-free, fast and easy to carry out.

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Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics

Cited by 38 publications

References 34 publications

Calculated free bilirubin levels and neurotoxicity

Calculated free bilirubin levels and neurotoxicity

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

Quantitative determination of bilirubin by inhibition of chemiluminescence from lucigenin

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