Article

Review: Bilirubin pKa studies: new models and theories indicate high pKa values in water, dimethylformamide and DMSO.

School of Pharmacy, University of Wisconsin, Madison, 53705-2222, USA.
BMC Biochemistry (Impact Factor: 1.78). 03/2010; 11:16. DOI: 10.1186/1471-2091-11-16
Source: PubMed

ABSTRACT Correct aqueous pKa values of unconjugated bilirubin (UCB), a poorly-soluble, unstable substance, are essential for understanding its functions. Our prior solvent partition studies, of unlabeled and [14C] UCB, indicated pKa values above 8.0. These high values were attributed to effects of internal H-bonding in UCB. Many earlier and subsequent studies have reported lower pKa values, some even below 5.0, which are often used to describe the behavior of UCB. We here review 18 published studies that assessed aqueous pKa values of UCB, critically evaluating their methodologies in relation to essential preconditions for valid pKa measurements (short-duration experiments with purified UCB below saturation and accounting for self-association of UCB).
These re-assessments identified major deficiencies that invalidate the results of all but our partition studies. New theoretical modeling of UCB titrations shows remarkable, unexpected effects of self-association, yielding falsely low pKa estimates, and provides some rationalization of the titration anomalies. The titration behavior reported for a soluble thioether conjugate of UCB at high aqueous concentrations is shown to be highly anomalous. Theoretical re-interpretations of data in DMSO and dimethylformamide show that those indirectly-derived aqueous pKa values are unacceptable, and indicate new, high average pKa values for UCB in non-aqueous media (>11 in DMSO and, probably, >10 in dimethylformamide).
No reliable aqueous pKa values of UCB are available for comparison with our partition-derived results. A companion paper shows that only the high pKa values can explain the pH-dependence of UCB binding to phospholipids, cyclodextrins, and alkyl-glycoside and bile salt micelles.

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    ABSTRACT: Correct aqueous pKa values of unconjugated bilirubin (UCB), a poorly-soluble, unstable substance, are essential for understanding its functions. Our prior solvent partition studies, of unlabeled and [14C] UCB, indicated pKa values above 8.0. These high values were attributed to effects of internal H-bonding in UCB. Many earlier and subsequent studies have reported lower pKa values, some even below 5.0, which are often used to describe the behavior of UCB. We here review 18 published studies that assessed aqueous pKa values of UCB, critically evaluating their methodologies in relation to essential preconditions for valid pKa measurements (short-duration experiments with purified UCB below saturation and accounting for self-association of UCB). These re-assessments identified major deficiencies that invalidate the results of all but our partition studies. New theoretical modeling of UCB titrations shows remarkable, unexpected effects of self-association, yielding falsely low pKa estimates, and provides some rationalization of the titration anomalies. The titration behavior reported for a soluble thioether conjugate of UCB at high aqueous concentrations is shown to be highly anomalous. Theoretical re-interpretations of data in DMSO and dimethylformamide show that those indirectly-derived aqueous pKa values are unacceptable, and indicate new, high average pKa values for UCB in non-aqueous media (>11 in DMSO and, probably, >10 in dimethylformamide). No reliable aqueous pKa values of UCB are available for comparison with our partition-derived results. A companion paper shows that only the high pKa values can explain the pH-dependence of UCB binding to phospholipids, cyclodextrins, and alkyl-glycoside and bile salt micelles.
    BMC Biochemistry 03/2010; 11:16. · 1.78 Impact Factor
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    ABSTRACT: Unconjugated bilirubin (UCB) is an unstable substance with very low aqueous solubility. Its aqueous pKa values affect many of its interactions, particularly their pH-dependence. A companion paper shows that only our prior solvent partition studies, leading to pKa values of 8.12 and 8.44, met all essential requirements for valid pKa determinations. Other published values, generally lower, some below 5.0, were shown to be invalid. The present work was designed to derive suitable models for interpreting published data on the pH-dependent binding of UCB with four agents, mentioned below, chosen because they are not, themselves, sensitive to changes in the pH range 4-10, and the data, mainly spectrometric, were of reasonable quality. These analyses indicated that the high pKa values, dianion dimerization constant and solubilities of UCB at various pH values, derived from our partition studies, along with literature-derived pH- and time-dependent supersaturation effects, were essential for constructing useful models that showed good qualitative, and sometimes quantitative, fits with the data. In contrast, published pKa values below 5.0 were highly incompatible with the data for all systems considered. The primary species of bound UCB in our models were: undissociated diacid for phosphatidylcholine, dianion for dodecyl maltoside micelles and cyclodextrins, and both monoanions and dianion for sodium taurocholate. The resulting binding versus pH profiles differed strikingly from each other. These analyses of UCB binding reinforce the evidence from UCB in simple systems that the pKa values of UCB are much higher than those of simple carboxylic acids. The insights derived from these analyses should be helpful to explore and interpret UCB binding to more complex, pH-sensitive, physiological moieties, such as proteins or membranes, in order to understand its functions.
    BMC Biochemistry 03/2010; 11:15. · 1.78 Impact Factor
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    ABSTRACT: Cerebrospinal fluid (CSF) was examined for bilirubin, an important indicator for diagnosis of subarachnoid hemorrhage (SAH). A multi-wavelength (340, 415, and 460 nm) spectrophotometric assay was developed for the quantitative measurement of bilirubin in CSF, enabling the mathematical correction for absorbance of hemoglobin and proteins. Bilirubin and hemoglobin results were correlated to HPLC and a standard colorimetric assay, respectively. A subset of samples were sent for an absorbance reading at 450 nm following baseline correction. The multi-wavelength bilirubin assay was validated on 70 patients with confirmed SAH and 70 patients with neurologic symptoms who ruled out for SAH. The multi-wavelength spectrophometric assay demonstrated no interferences due to proteins (albumin) up to 30 g/l or oxyhemoglobin up to 260 mg/l. The assay limit of detection was 0.2 mg/l, linear to 20 mg/l, and CVs ranged from 1-6% at bilirubin concentrations of 0.84 and 2.1 mg/l. The spectrophotometric assay correlated to HPLC and the colorimetric assay for bilirubin and hemoglobin, respectively. Results also correlated to the absorbance method (with removal of samples with high hemoglobin and proteins). The area under the ROC curve for diagnosis of SAH was 0.971 and 0.954 for the HPLC and spectrophotometric assay, respectively. At a cutoff of 0.2 mg/l, the clinical specificity was 100% for both assays, and the clinical sensitivity was 94.3% and 88.6% for SAH for the HPLC and spectrophotometric asays, respectively. The multi-wavelength spectrophotometric assay is an objective alternative to visual inspection, HPLC, and absorbance for CSF bilirubin.
    Clinica chimica acta; international journal of clinical chemistry 06/2013; · 2.54 Impact Factor

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