The Preparation of Crystalline Bilirubin-C14*

Ostrow, J. Donald; Hammaker, Lydia; Schmid, Rudi

doi:10.1172/jci104375

Cited by 195 publications

(114 citation statements)

References 50 publications

(50 reference statements)

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“…The formation of bilirubin as the end product of heme oxygenase activity was confirmed by extraction of the incubation mixtures with chloroform and crystallization (7) and spectral analysis of the bile pigment. The concentration of hematin routinely used was 17 ,uM, except where noted.…”

Section: Methodsmentioning

confidence: 90%

Cobalt Induction of Hepatic Heme Oxygenase; with Evidence That Cytochrome P -450 Is Not Essential for This Enzyme Activity

Maines

Kappas

1974

Proc. Natl. Acad. Sci. U.S.A.

244

129

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Treatment of rats in vivo with cobalt chloride stimulated heme oxidation by hepatic microsomes to levels up to 800% above controls. This treatment also caused increases in liver weight and in total microsomal protein; in contrast, marked decreases were produced in microsomal oxidation of ethylmorphine (80%), and in cytochrome. P-450 (60-70%) and heme (30-50%) contents. Cobalt chloride treatment did not affect heme oxidation by the spleen heme oxygenase system.The rate of heme oxidation by hepatic microsomal enzymes and the microsomal content of cytochrome P-450 were found to be unrelated. This conclusion was reached from studies in which microsomal heme oxygenase activity from cobalt-treated animals could be increased by 900% above control levels in the same microsomal preparation in which cytochrome P-450 content was decreased to spectrally unmeasurable amounts after incubation with 4 M urea. The same treatment eliminated ethylmorphine demethylation and decreased microsomal NADPH-cytochrome c reductase (EC 1.6.2.4) activity by 75%.It is concluded that (i) the hepatic microsomal enzyme system that oxidizes heme compounds is not the same as that which metabolizes drugs, (ii) cytochrome P-450 is not essential for the oxidation of heme by liver cells, (iii) there is no direct relationship between the rate of heme oxidation and the level of NADPH-cytochrome c reductase activity, and (iv) the oxidation of heme is protein-dependent and the active proteins are inducible, but are different from those involved in drug metabolism.

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Section: Methodsmentioning

confidence: 90%

Cobalt Induction of Hepatic Heme Oxygenase; with Evidence That Cytochrome P -450 Is Not Essential for This Enzyme Activity

Maines

Kappas

1974

Proc. Natl. Acad. Sci. U.S.A.

244

129

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“…Bilirubin (Sigma) was recrystallized from chloroform-methanol according to the procedure of Ostrow et al (33). Purity was judged to be adequate by virtue of spectrophotometric extinction coefficients in agreement with reported values (14).…”

Section: Methodsmentioning

confidence: 67%

Bilirubin Binding in the Plasma of Newborns: Critical Evaluation of a Fluorescence Quenching Method and Comparison to the Peroxidase Method

et al. 1984

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SummaryBilirubin binding affinities and capacities and apparent unbound ("free") bilirubin levels were determined in serum samples from 47 high-risk newborns, in 22 samples of cord serum, and in serum samples from 15 Greek children with marked hyperbilirubinemia, by both fluorescence quenching and peroxidase methods. The free fatty acid:albumin molar ratio was also determined for serum samples from high-risk newborns. In vitro and in vivo measurements suggest that free fatty acids are rarely present at levels that produce significant displacement of bilirubin, which is in agreement with previous studies. The two bilirubin binding assays showed only fair correlation with sizable discrepancies for many specimens. Technical difficulties inherent in the fluorescence quenching method and possible sources of error are discussed. Our observations suggest that routine application of these two assays as the primary criterion for therapeutic intervention (e.g., exchange transfusion) is premature.

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“…Purified 14 C-unconjugated bilirubin was then obtained by a modification of the method of Ostrow et al (21). Briefly, proteins were removed from pooled bile using reverse-phase C18 cartridges (Mega Bond Elut, Analytichem, Harbor City, CA).…”

Section: Methodsmentioning

confidence: 99%

“…After extraction into chloroform, the labeled bilirubin was further purified by two cycles of alkaline extraction followed by recrystallization to constant specific activity according to McDonagh and Assisi (22). The resulting [ 14 C]bilirubin had a specific radioactivity of 5 ϫ 10 4 dpm/g or 0.022 Ci/g, as assessed by scintillation counting and diazo assay (23), and met criteria for purity described previously (21). The [ 14 C]bilirubin solution was distributed among glass vials, evaporated to dryness, sealed under argon, and finally stored in the dark at Ϫ20°C until use (18 (23) were performed in duplicate on 0.5 ml of the solution, and the remaining 2.5 ml was used immediately for ultrafiltration studies.…”

Section: Methodsmentioning

confidence: 99%

Affinity of Human Serum Albumin for Bilirubin Varies with Albumin Concentration and Buffer Composition

Weisiger¹,

Ostrow²,

Koehler³

et al. 2001

Journal of Biological Chemistry

Self Cite

104

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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 [ 14 C]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 [ 14 C]bilirubin was strongly dependent on assay conditions, falling from (5.09 ؎ 0.24) ؋ 10 7 liters/mol at lower albumin concentrations (15 M) to (0.54 ؎ 0.05) ؋ 10 7 liters/mol at higher albumin concentrations (300 M). 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) ؋ 10 7 liters/mol to (0.65 ؎ 0.03) ؋ 10 7 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.

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The Preparation of Crystalline Bilirubin-C14*

Cited by 195 publications

References 50 publications

Cobalt Induction of Hepatic Heme Oxygenase; with Evidence That Cytochrome P -450 Is Not Essential for This Enzyme Activity

Cobalt Induction of Hepatic Heme Oxygenase; with Evidence That Cytochrome P -450 Is Not Essential for This Enzyme Activity

Bilirubin Binding in the Plasma of Newborns: Critical Evaluation of a Fluorescence Quenching Method and Comparison to the Peroxidase Method

Affinity of Human Serum Albumin for Bilirubin Varies with Albumin Concentration and Buffer Composition

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