Radioassay of UDP-glucuronyltransferase-catalysed formation of bilirubin monoglucuronides and bilirubin diglucuronide in liver microsomes

Vermeir, Marc; Vanstapel, Florent; Blanckaert, Norbert

doi:10.1042/bj2230455

Cited by 13 publications

(10 citation statements)

References 11 publications

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“…4.8 zero-order kinetic conditions the diglucuronide amounted to 22-23% of the rate of formation of the total BMG (Table 1). These results are in agreement with those obtained in previous work (Blanckaert et al, 1979;Gordon et al, 1983;Vermeir et al, 1984). Further, the initial rates of formation of the two isomeric (C-8, C-12) BMGs by these microsomal preparations indicated that the C-8 isomer was produced approximately twice as rapidly as the C-12 isomer (Table 1), confirming previous work of Blanckaert et al (1979).…”

Section: Results and Discussion Enzyme Purification And Bilirubin Glusupporting

confidence: 92%

“…Chemicals and reagents were as described elsewhere (Burchell, 1977(Burchell, , 1980; the preceding paper, Vermeir et al, 1984), except when stated otherwise in the text. The UDP-sugars were purchased from Sigma (St. Louis, MO, U.S.A.).…”

Section: Methodsmentioning

confidence: 99%

“…Radioassay of initial rates of formation of the individual C-8 and C-12 BMGs and BDG was performed with [14C]bilirubin as substrate and with alkaline methanolysis combined with t.l.c. for analysis of the reaction products (Vermeir et al, 1984). Incubation mixtures contained 10 or 50pM-bilirubin, lOmM-UDPglucuronic acid, 2mM-UDP-N-acetylglucosamine and 1mg of microsomal protein/ml.…”

Section: Methodsmentioning

confidence: 99%

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Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase

Burchell¹,

Blanckaert²

1984

Biochemical Journal

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Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.

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Section: Results and Discussion Enzyme Purification And Bilirubin Glusupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase

Burchell¹,

Blanckaert²

1984

Biochemical Journal

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“…9–11 Radioassays have been used to quantify antigens at sub-pM concentrations, drugs such as buprenorphine at sub-nM concentrations, 1, 8 and enzyme activity. 4–5…”

Section: Introductionmentioning

confidence: 99%

“…Radionuclides, specifically 3 H, 33 P, and 35 S (all β-particle emitters), are commonly used as labels in biological assays due to the distribution of these atoms in biological systems. − Unlike fluorophores or fluorescent protein tags, radionuclides do not significantly increase the size or mass of the labeled component and therefore have minimal effects on binding, conformational changes, diffusion and active transport, etc. In addition, lower backgrounds are typically obtained for radioassays compared to fluorescence assays, where background arises from the inherent fluorescence of many species within the sample. − Radioassays have been used to quantify antigens at sub-pM concentrations, drugs such as buprenorphine at sub-nM concentrations, , and enzyme activity. , …”

Section: Introductionmentioning

confidence: 99%

Polystyrene-Core, Silica-Shell Scintillant Nanoparticles for Low-Energy Radionuclide Quantification in Aqueous Media

Janczak

Calderon

Mokhtari

et al. 2018

ACS Appl. Mater. Interfaces

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β-particle emitting radionuclides are useful molecular labels due to their abundance in biomolecules. Detection of β-emission from H,S, and P, important biological isotopes, is challenging due to the low energies (E ≤ 300 keV) and short penetration depths (≤0.6 mm) in aqueous media. The activity of biologically relevant β-emitters is usually measured in liquid scintillation cocktail (LSC), a mixture of energy-absorbing organic solvents, surfactants, and scintillant fluorophores, which places significant limitations on the ability to acquire time-resolved measurements directly in aqueous biological systems. As an alternative to LSC, we developed polystyrene-core, silica-shell nanoparticle scintillators (referred to as nanoSCINT) for quantification of low-energy β-particle emitting radionuclides directly in aqueous solutions. The polystyrene acts as an absorber for energy from emitted β-particles and can be loaded with a range of hydrophobic scintillant fluorophores, leading to photon emission at visible wavelengths. The silica shell serves as a hydrophilic shield for the polystyrene core, enabling dispersion in aqueous media and providing better compatibility with water-soluble analytes. While polymer and inorganic scintillating microparticles are commercially available, their large size and/or high density complicates effective dispersion throughout the sample volume. In this work, nanoSCINT nanoparticles were prepared and characterized. nanoSCINT responds to H,S, and P directly in aqueous solutions, does not exhibit a change in scintillation response between pH 3.0 and 9.5 or with 100 mM NaCl, and can be recovered and reused for activity measurements in bulk aqueous samples, demonstrating the potential for reduced production of LSC waste and reduced total waste volume during radionuclide quantification. The limits of detection for 1 mg/mL nanoSCINT are 130 nCi/mL forH, 8 nCi/mL for S, and<1 nCi/mL for P.

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Thyroid hormones and the hepatic handling of bilirubin. I. Effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the wistar rat

et al. 1989

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The effects of thyroidectomy and of thyroid hormone administration on the hepatic transport of endogenous bilirubin were investigated in the Wistar R/APfd rat. Hypothyroidism resulted in an enhanced hepatic bilirubin UDP-glucuronosyltransferase activity and in a decreased p-nitrophenol transferase activity. It caused a cholestatic condition with a 50% decrease in bile flow and bile salt excretion, and an increased proportion of conjugated bilirubin in serum. The biliary output of unconjugated and monoconjugated bilirubins decreased in parallel by about 65%, whereas the excretion rate of the diconjugate dropped by only 47%, resulting in an increased di- to monoconjugate ratio in bile. Hyperthyroidism was characterized by a decreased bilirubin and an increased p-nitrophenol transferase activity, and by an augmented bilirubin output in bile. The output of unconjugated and monoconjugated bilirubins increased in parallel by about 50 or 100%, whereas the excretion of the diconjugate increased by only 20 to 50%, depending on the dose of thyroxine administered; this resulted in a decreased di- to monoconjugate ratio in bile. A linear positive relationship was found between bilirubin UDP-glucuronosyltransferase activity and the ratio of bilirubin di- to monoconjugates present in bile or formed by in vitro incubation of liver homogenates at low concentration of bilirubin (10 to 15 microM), indicating that bile pigment composition is mainly determined by the conjugation activity in the liver. The inverse relationship observed between hepatic beta-glucuronidase activity and the ratio of di- to monoconjugates in bile warrants further investigation to analyze whether this enzyme activity also plays a possible role in the changes in bile pigment composition in hypo- and hyperthyroid rats.

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Radioassay of UDP-glucuronyltransferase-catalysed formation of bilirubin monoglucuronides and bilirubin diglucuronide in liver microsomes

Cited by 13 publications

References 11 publications

Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase

Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase

Polystyrene-Core, Silica-Shell Scintillant Nanoparticles for Low-Energy Radionuclide Quantification in Aqueous Media

Thyroid hormones and the hepatic handling of bilirubin. I. Effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the wistar rat

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