Cecile Webster

Università degli Studi di Trieste, Trst, Friuli Venezia Giulia, Italy

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Publications (5)25.9 Total impact

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    ABSTRACT: We report problems encountered during preparation of tritium-labeled unconjugated bilirubin ((3)H-UCB) from precursor (3)H-5-aminolevulinic acid ((3)H-ALA) in 2 dogs with external biliary drainage installed into the animals under general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCi of (3)H-ALA was administered intravenously in two divided doses, and bile was collected for 9 hours. In one animal, taurocholate (TC) infusion was needed to maintain bile flow. (3)H-UCB was isolated from the bile and recrystallized with the improved method of Webster et al (Webster CC, Tiribelli C, Ostrow JD. J Lab Clin Med 2001;137:370-3). Based on radioactivity and pigment content, hourly bile collections were pooled to optimize specific activities. Surprisingly, in the first dog, only 2.9% of injected radioactivity was recovered in bile and only 14.1% in urine, and the specific activities of the crystalline (3)H-UCB from the two pools were only 39.5 and 30.0 x 10(3) dpm/microg. High-performance liquid chromatography analysis revealed that only 4% of ALA degraded during 5 minutes in injection solution at pH 6.8. The low incorporation of (3)H-ALA and low specific activity of (3)H-UCB was apparently caused mainly by prior degradation and exchange of labile tritium of the (3)H-ALA and probably by enhanced endogenous ALA synthesis caused by the anesthetic/sedative agents. Revised procedures in the second dog improved the incorporation of (3)H-ALA to 11.9% excreted in bile and the specific activity of the crystalline (3)H-UCB to 122.0 and 50.8 x 10(3) dpm/microg, while urinary excretion of tritium increased to 28.5%. These experiences emphasize possible pitfalls in preparing (3)H-UCB by biosynthetic labeling from (3)H-ALA administered to dogs.
    Full-text · Article · Dec 2001 · Journal of Laboratory and Clinical Medicine
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    ABSTRACT: Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [14C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [14C]bilirubin was strongly dependent on assay conditions, falling from (5.09 ± 0.24) × 107liters/mol at lower albumin concentrations (15 μm) to (0.54 ± 0.05) × 107 liters/mol at higher albumin concentrations (300 μm). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11–0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 ± 0.26) × 107 liters/mol to (0.65 ± 0.03) × 107 liters/mol). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.
    Full-text · Article · Sep 2001 · Journal of Biological Chemistry
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    Cecile Webster · Claudio Tiribelli · J.Donald Ostrow
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    ABSTRACT: Radiolabeled unconjugated bilirubin (UCB) is currently prepared by biosynthetic labeling of bilirubin in fistula bile from precursor-labeled delta-aminolevulinic acid (ALA) in rats or dogs. With existing methods, yields of labeled UCB from the bile are generally less than 50%. We here report modifications of the original method of Ostrow et al (Ostrow JD, Hammaker L, Schmid R. The preparation of crystalline bilirubin-C(14). J Clin Invest 1961;40:1442-52) that result in improvement of yields to 72% from both dog and rat bile. The modifications include the initial deproteination of bile with a reverse-phase C18 cartridge, removal of ethanol before alkaline hydro-lysis to avoid esterification of UCB, and adjustments for the high proportion of non-glucuronide UCB conjugates in dog bile not precipitated as lead salts. These improvements should save significantly on both costs and animal usage.
    Full-text · Article · Jun 2001 · Journal of Laboratory and Clinical Medicine

  • No preview · Article · Dec 2000 · Journal of Hepatology
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    ABSTRACT: Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.
    Full-text · Article · Jul 1996 · Biochemical Journal

Publication Stats

134 Citations
25.90 Total Impact Points


  • 2001
    • Università degli Studi di Trieste
      • Center for the Study of Liver Disease
      Trst, Friuli Venezia Giulia, Italy