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

1984 ◽  
Vol 223 (2) ◽  
pp. 455-459 ◽  
Author(s):  
M Vermeir ◽  
F Vanstapel ◽  
N Blanckaert
Keyword(s):  
Enzyme Assay ◽  
Total Bilirubin ◽  
Glucuronic Acid ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Reaction Products ◽  
Accuracy And Precision ◽  
Specific Determination ◽  
Alkaline Methanolysis

A radioassay for specific determination of the rates of UDP-glucuronic acid-dependent conversion of bilirubin into the two isomeric (C-8, C-12) bilirubin monoglucuronides and bilirubin diglucuronide is described and illustrated by its application to rat liver microsomes. The method is based on measurement of the relative amounts of radiolabel in unesterified bilirubin and its mono- and di-esterified reaction products after incubation with [14C]bilirubin as substrate. This analysis is performed by the alkaline-methanolysis procedure, combined with one of two t.l.c. systems developed in order to enhance the sensitivity, accuracy and precision of the radioassay. Results for rates of total bilirubin glucuronide formation obtained with the new assay and the standard enzyme assay based on the ethyl anthranilate diazo-method were identical. However, the sensitivity of the latter technique is approx. 10-fold lower than that of the radioassay.

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

1984 ◽  
Vol 223 (2) ◽  
pp. 461-465 ◽  
Author(s):  
B Burchell ◽  
N Blanckaert
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Total Bilirubin ◽  
Glucuronic Acid ◽  
Liver Microsomes ◽  
Wistar Rat ◽  
Zero Order ◽  
Rat Liver Microsomes ◽  
Order Kinetic ◽  
Hepatic Microsomes

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.

Determination of metabolic profile of anti-malarial trioxane CDRI 99/411 in rat liver microsomes using HPLC

2011 ◽  
Vol 26 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Smriti Mishra ◽  
Lakshmi Manickavasagam ◽  
Girish Kumar Jain
Keyword(s):  
Rat Liver ◽  
Metabolic Profile ◽  
Liver Microsomes ◽  
Rat Liver Microsomes

scholarly journals The effect of steroids and nucleotides on solubilized bilirubin uridine diphosphate glucuronyltransferase

1970 ◽  
Vol 119 (3) ◽  
pp. 437-445 ◽  
Author(s):  
B. P. F. Adlard ◽  
G. H. Lathe
Keyword(s):  
Enzyme Activity ◽  
Glucuronic Acid ◽  
Liver Microsomes ◽  
Fold Increase ◽  
Rat Liver Microsomes ◽  
Uridine Diphosphate ◽  
Competitive Effects ◽  
Solubilized Enzyme ◽  
High Concentrations ◽  
Solubilized Form

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis–Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The Km for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca2+ were inhibitory in the absence of Mg2+ but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3β-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.

A Direct, Highly Sensitive Fluorometric Assay for a Microsomal Cytochrome P450-Mediated O-Demethylation Using a Novel Coumarin Analog as Substrate

2000 ◽  
Vol 55 (11-12) ◽  
pp. 915-922 ◽  
Author(s):  
René Roser ◽  
Helmut Thomas
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Fluorometric Assay ◽  
Excitation Wavelength ◽  
Monooxygenase System ◽  
Rat Liver Microsomes ◽  
Monooxygenase Activity ◽  
Continuous Increase ◽  
Highly Sensitive

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (λA = 380 nm, λF = 480 nm ) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 480 nm and a fluorescence/em ission w avelength of 480 nm, the fluorescence of this substance (λA = 338 nm, λF = 422 nm ) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrom e P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.

An Automated Method for the Determination of Serum Bilirubin

1964 ◽  
Vol 10 (5) ◽  
pp. 399-405 ◽  
Author(s):  
Murray Golub
Keyword(s):  
Chemical Analysis ◽  
Good Accuracy ◽  
Total Bilirubin ◽  
Serum Bilirubin ◽  
Diazonium Salt ◽  
Reference Method ◽  
Precision Data ◽  
Automated Method ◽  
Accuracy And Precision

Abstract The bilirubin method of Rand and DiPasqua (3), which utilizes a stabilized diazonium salt solution of 2,4 dichloraniline, has been adapted to automatic chemical analysis. Both total bilirubin and "1-min. reacting" bilirubin can be assayed with good accuracy and precision. Data are presented to show excellent agreement with the manually performed procedure and the procedure of Malloy and Evelyn (1), which was used as the reference method.

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